Download Protein-protein interactions: mechanisms and

JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2002; 15: 405–422
Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.597
Protein–protein interactions: mechanisms and
modification by drugs
A. V. Veselovsky1*, Yu. D. Ivanov1, A. S. Ivanov1, A. I. Archakov1, P. Lewi2 and P. Janssen2
1
Institute of Biomedical Chemistry, Moscow, Russia
Center for Molecular Design, Janssen Pharmaceutica N.Y., Belgium
2
Protein–protein interactions form the proteinaceous network, which plays a central role in numerous
processes in the cell. This review highlights the main structures, properties of contact surfaces, and forces
involved in protein–protein interactions. The properties of protein contact surfaces depend on their
functions. The characteristics of contact surfaces of short-lived protein complexes share some similarities
with the active sites of enzymes. The contact surfaces of permanent complexes resemble domain contacts or
the protein core. It is reasonable to consider protein–protein complex formation as a continuation of protein
folding. The contact surfaces of the protein complexes have unique structure and properties, so they
represent prospective targets for a new generation of drugs. During the last decade, numerous investigations
have been undertaken to find or design small molecules that block protein dimerization or protein(peptide)–
receptor interaction, or on the other hand, induce protein dimerization. Copyright # 2002 John Wiley &
Sons, Ltd.
Keywords: protein–protein interaction; protein surface; inhibitors; drug design; antagonist; protein complex;
thermodynamics; dimerization
Received 8 Junuary 2002; revised 18 March 2002; accepted 28 June 2002
INTRODUCTION
Protein–protein interaction is a common mechanism
responsible for functioning of numerous processes in the
cell. Protein complex formation is crucial for formation of
active sites of oligomer enzymes and maintenance of their
effective conformation (Banci et al., 1998; Holwerda,
1999). It is also crucial for numerous regulatory processes,
including signal transduction (Eyster, 1998; Klemm et al.,
1998; Souroujon and Mochly-Rosen, 1998), cell–cell
contacts (Alattia et al., 1999), electron transport systems
(respiratory chain, cytochrome P450 oxygenase system;
Cunha et al., 1999; Schenkman and Jansson, 1999),
antigen–antibody interaction (Dall’Acqua et al., 1998;
Salzmann and Bachmann, 1998), DNA synthesis (Dear et
al., 1997; Sengchanthalangsy et al., 1999), formation of
intracellular structures (Herrmann and Aebi, 1998; Hilpert
et al., 1999) etc. Pathological protein complex formation
may be responsible for the development of some components of Alzheimer’s and prion diseases (Cohen and
Prusiner, 1998; Selkoe, 1998). Furthermore, protein aggregation may occur as a result of protein extraction and
subsequent protein purification; this is typical, especially for
membrane proteins (Kiselyova et al., 1999). In these cases,
proteins bind each other rather unspecifically and this leads
*Correspondence to: A. V. Veselovsky, Institute of Biomedical Chemistry
RAMS, Pogodinskaya str. 10, Moscow 119992, Russia.
E-mail: [email protected]
Contract/grant sponsor: RFBR; contract/grant number: N 99-04-48081;
contract/grant number: N 01-04-48128.
Abbreviations used: HIV, human immunodeficiency virus; hGH, human
growth hormone; ID, inhibitor of dimerization; PH, pleckstrin homology; SH2,
Src-homology-2; SH3, Src-homology-3.
Copyright # 2002 John Wiley & Sons, Ltd.
to artificial generation of protein complexes with irregular
structure.
Depending on the stability and mechanism of protein–
protein complex formation protein complexes can be
subdivided into non-obligate (short-living) complexes and
permanent stable complexes (proteins are native only in
oligomeric structures; Jones and Thornton, 1996, 2000). Tsai
et al. (1997b) proposed two-state and three-state models for
such complex formation. According to the former model,
contacting proteins can ‘exist either unfolded or folded
together in a complex’, whereas the three-state model of
complexes implies that each protein folds separately and
only after that can folded proteins form the complex (Tsai et
al., 1997b). So the formation of the permanent complexes
can be considered as a continuation of protein folding. It is
reasonable to suggest that the structure and properties of the
interfaces of the proteins forming these two types of
complexes must differ in their structure and properties.
Currently, a huge amount of information about various
aspects of protein–protein interactions has been accumulated. In this review, we consider the common mechanisms
underlying physiological protein–protein interactions and
recent achievements in the design of compounds regulating
complex formation or disintegrating pre-existing complexes. These studies culminate in the development of a
new class of biologically active low-weight non-peptide
compounds modifying protein–protein interactions.
STRUCTURE AND PROPERTIES OF
PROTEIN–PROTEIN CONTACTS
Most information about protein contact areas was obtained
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A. V. VESELOVSKY ET AL.
from analysis of three-dimensional structures solved by
X-ray crystallography or NMR. Some useful information
was obtained using mutagenic screening (Bogan and Thorn,
1998; Dall’Acqua et al., 1998; Massova and Kollman,
1999), site-directed mutagenesis (Martin et al., 1999b; Ortiz
et al., 1999; Otzen and Fersht, 1999; Vaughan et al., 1999)
and chemical modification (Fancy and Kodadek, 1999;
Ubarretxena-Belandia et al., 1999). Other methods were
also employed for analysis of protein–protein contacts.
They included fluorescent methods (Bhattacharya et al.,
1996; Park and Raines, 1997; Sloan and Hellinga, 1998),
calorimetric analysis (Aoki et al., 1998a; Leavitt, Freire,
2001), the two-hybrid system (Hu et al., 2000a), biosensor
methods (Glaser and Hausdorf, 1996; Ivanov et al., 1999a,b,
2001; McDonnell, 2001; Rich and Myszka, 2001) etc.
(Appling, 1999; Beeckmans, 1999; Ehring, 1999; Kameshita et al., 1998; Rudert et al., 1998; Rudiger et al., 1999;
Velev et al., 1998; Vergnon and Chu, 1999; Viani et al.,
2000).
The shape of the protein interface
The contact surface area consists of 6–30% of the monomer
surface area and may vary from 550 to 4900 Å2. The
average value of the contact surface of monomers is about
800 Å2 (Jones and Thornton, 1996; Stites, 1997). There is a
poor correlation between solvent accessible surface area of
the permanent complex and its molecular weight. No
correlation was found for non-obligate complexes, although
their interfaces were more planar (Jones and Thornton,
1996).
Amino acid composition
Repeated attempts were undertaken to determine possible
enrichment of amino acid residues in the protein contact
interfaces with certain amino acids. Some authors detected
an increased number of arginine, histidine, asparagine,
tryptophan, tyrosine and serine residues in the contact
regions in comparison with their common content in the
protein (Stites, 1997). Some authors found increased content
of aromatic amino acids (Davies and Cohen, 1996) or
hydrophobic amino acid residues (Jones and Thornton,
1996; Ivanov et al., 1999a, 2000). Such a variety of the data
may reflect different sets of protein complexes used for the
analysis. Another reason for such diversity may be the
different nature of the analysed complexes. Since the
contact interfaces of permanent complexes are similar to the
protein core (Tsai et al., 1996, 1997a), the hydrophobic
amino acids apparently predominate (Jones and Thornton,
1996; Stites, 1997). However, in non-obligate complexes,
where contact with the aqueous environment is quite
possible, hydrophilic and charged amino acids may
predominate (Jones and Thornton, 1996; Ivanov et al.,
1999b, 2001). So it seems unlikely that the contact interface
is actually characterized by an increased proportion of
certain amino acids. It is possible that the prevalence of
some amino acids in certain contact surfaces reflects the
specific properties of protein areas involved in these
interactions.
Copyright # 2002 John Wiley & Sons, Ltd.
Secondary structure
All types of secondary structure (helices, beta-sheets, turns
and random coil) have been found in contact areas of the
interacting proteins (Stites, 1997; Tsai et al., 1997a).
Analysis of 225 complexes revealed the following range
of distribution of the secondary structures in interface areas:
random coil (47%) >a-helix (36%) >b-sheet (17%). The
distribution of the second structures in contact interfaces
depends on the type of the complex formed (permanent or
non-obligate; Jones and Thornton, 1996). The architecture
of the permanent complex interfaces is similar to the protein
core, exhibiting limited sets of protein folding patterns. The
distribution of secondary structures in the interface of nonobligate complexes shows larger variability and resembles
exterior protein surfaces with the exception of a higher ratio
of helices (Tsai et al., 1997a). Usually contact area
represents short segments of the secondary structures. The
protein contact interfaces include from 1 to 15 such
segments (Jones and Thornton, 1996). In most cases, the
interface surfaces consist of various types of secondary
structure, but a single type of secondary structure was also
found there (Jones and Thornton, 1996).
Frequently various proteins involved in protein complex
formation have stable structural domains denominated with
their own titles. They include helix–loop–helix domains
(Ghosh and Chmielewski, 1998; Norton et al., 1998), SH2
(Src-homology-2) (Pawson, 1995; Pawson et al., 2001),
SH3 (Src-homology-3; Pawson, 1995), PH (pleckstrin
homology; Pawson, 1995), PDZ (Songyang et al., 1997)
and PDZ2 (Kozlov et al., 2000) domains and others (Blatch
and Lassle, 1999; Schumacher et al., 2000; Zhang et al.,
1998; Fig. 1). Similar stable structural domains in different
proteins can participate in the regulation of various cell
processes (Ahmad et al., 1998; Zhang et al., 1998). These
domains recognize and bind to certain short peptide motifs.
For example, the SH3 domain recognizes a proline-rich
motif (Pawson, 1995). One protein with such a domain can
bind several different proteins (Gotz et al., 1999; Foti et al.,
1999; Onofri et al., 2000; Souroujon and Mochly-Rosen,
1998) or one protein can contain several such domains
(Dobrosotskaya et al., 1997; Dong et al., 1998).
Forces involved in protein–protein interactions
Steric, hydrophobic, electrostatic interactions and hydrogen
bonds are the main factors responsible for protein–protein
interactions.
(a) Steric complementarity. Analysis of protein contacts
revealed that their interface surfaces are quite complementary to each other (Jones and Thornton, 1996; Stites, 1997;
Tsai et al., 1997b). The degree of complementarity depends
on the type of protein interaction. The permanent complexes
exhibit highest complementarity. Non-obligate complexes
and protein–inhibitor complexes are characterized by lower
complementarity, and antigen–antibody complexes have the
worst complementarity (Jones and Thornton, 1996; Stites,
1997; Tsai et al., 1997b). Usually protein interfaces in
protein complexes contain some cavities. Analysis of the
interface surfaces of 24 intersubunit contacts showed that
J. Mol. Recognit. 2002; 15: 405–422
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
407
Figure 1. Some examples of structure of stable domains with their interacting peptides. (A) PDZ domain from
neuronal nitric oxide 2 synthase (PDB1B8Q); (B) SH2 domain from phosphotransferase (PDB1SPS); (C) SH3
domain from FYN proto-oncogene 2 tyrosine kinase (PDB1A0N). Interacting peptides are shown as sticks.
only two of them do not have such cavities. Usually cavity
surfaces represent about 10% of total interface surfaces.
Most cavities (about 63%) are filled with solvent (Hubbard
and Argos, 1994).
(b) Hydrophobic interaction. The important contribution
of the hydrophobic force to the protein–protein interaction
has been demonstrated in numerous studies (Eisenhaber and
Argos, 1996; Tsai et al., 1996, 1997a; Wells, 1996). The
average values of the hydrophobicity of contact surfaces
usually represent a mean of the hydrophobicity of the
protein core and its surface (Jones and Thornton, 1996; Tsai
et al., 1997a). The contribution of the hydrophobic
interaction is higher in permanent complexes than in nonobligate complexes (Jones and Thornton, 1996). The latter
can be explained by the fact that permanent complexes
usually exist in the bound state and the hydrophobic force is
more preferential for this purpose, whereas non-obligate
complexes are assembled in the water environment for
rather a short time, and this makes energetically unfavourable the high hydrophobicity of their surfaces. However the
non-obligate complexes of membrane proteins, such as
cytochrome P450 2B4, in contrast to water-soluble proteins,
are formed by hydrophobic interaction of their membrane
parts (Ivanov et al., 1999a, 2000). In the case of enzyme
interaction with peptide inhibitors or substrates, the
contacted interfaces may have hydrophilic surfaces (Jones
and Thornton, 1996; Stevens et al., 2000).
The hydrophobic regions in the contact interfaces are
organized as patches. The number of such patches may vary
from 1 to 15. Usually their sizes are within 200–400 Å2, but
they can achieve 3000 Å2 (Lijnzaad and Argos, 1997).
Analysis of complementarity of the large patches of
interfaces from different subunits showed their low overlap
of each other (Lijnzaad and Argos, 1997).
(c) Electrostatics. The electrostatic force is the other
significance force involved in protein–protein interactions
(Gong et al., 2000; Grucza et al., 2000; Muegge et al., 1998;
Sheinerman et al., 2000; Stevens et al., 2000; Xu et al.,
1997a; Zeng et al., 1999; Ivanov et al., 2001). Originally it
was assumed that the charges on the contacting surfaces are
located complementarily to each other; however, the
modern viewpoint suggests the electrostatic complementarity of interacting protein surfaces (McCoy et al., 1997).
Copyright # 2002 John Wiley & Sons, Ltd.
The charge density varies from 0 to 12 charged groups per
interface surface (Xu et al., 1997b). The distribution of the
opposite charges in the interfaces of the contacting area
showed that salt bridges across them are highly favourable
(Drozdov-Tikhomirov et al., 2001; Xu et al., 1997a,b). The
desolvation cost of the charged groups in salt-links is lower,
since they have favourable interactions with other charges
and hydrophilic residues surrounding them (Xu et al.,
1997a). It was proposed that a long-range attractive
electrostatic force could promote formation of encounter
complexes and therefore accelerate the rate of complex
formation (for review see Gabdoulline & Wade, 1999). Also
the electrostatic interaction can define the lifetime of
complexes (Archakov and Ivanov, 1999).
(d) Hydrogen bonding. The average number of hydrogen
bonds is proportional to the area of subunit interfaces: one
bond for each 100–200 Å2 (Jones and Thornton, 1996) or
about 10 bonds per interface (Lo Conte et al., 1999; Xu et
al., 1997b). The hydrogen bonds are preferably of oxygen–
nitrogen type (Xu et al., 1997b). The major proportion of the
hydrogen bonds is formed by side chains of amino acids
(about 76% of all hydrogen bonds). The exceptions are bsheet interfaces as for HIV protease (Wlodawer and
Vondrasek, 1998), or protein complexes with peptide
inhibitor or substrate (Jones and Thornton, 1996), where
the groups of the main chain usually form hydrogen bonds.
However, the hydrogen bonds in protein interfaces are
usually not in the optimal position, so they ‘are normal or
weak in terms of energetics’ (Xu et al., 1997b). Some
hydrogen bonds are formed between protein contact
surfaces and water molecules located near them (Tsai et
al., 1996; Xu et al., 1997b). Contrary to hydrogen bonds
formed between protein surfaces, the protein–water hydrogen bonds are ‘good’ ones (Xu et al., 1997b). Since water
molecules form more than one hydrogen bond they can
interact with a protein group and with another water
molecule, forming a network in protein–protein interfaces
(Davies and Cohen, 1996; Janin, 1999; Xu et al., 1997b).
(e) Water in interfaces. Water molecules are frequently
present at the complex interfaces (Davies and Cohen, 1996;
Janin, 1999; Wells, 1996). The number of the water
molecules usually varies from 1 to 50 (Davies and Cohen,
1996). Water molecules surround the contacting interfaces
J. Mol. Recognit. 2002; 15: 405–422
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A. V. VESELOVSKY ET AL.
or are buried in them (Davies and Cohen, 1996; Larsen et
al., 1998). In the latter case, they are located in the cavities
of the protein interfaces (Dall’Acqua et al., 1998; Vaughan
et al., 1999). The water molecules in the cavities may be
highly coordinated (Pardanani et al., 1998). They form
hydrogen bonds with protein groups and other water
molecules, and results this in aqueous networks along the
protein interfaces (Dall’Acqua et al., 1998; Janin, 1999; Xu
et al., 1997b). Interface water molecules stabilize the
protein complexes by forming additional hydrogen bonds,
by interacting with charges, and by increasing shape and
charge complementarity (Janin, 1999; Larsen et al., 1998;
Li et al., 2000; Pardanani et al., 1998; Xu et al., 1997b).
Protein–protein binding is accompanied by partial desolvation of the contacted surfaces and this predominates in
complexes in which one of reactants is neutral or weakly
charged (Camacho et al., 1999, 2000).
(f) Conformation. In some cases, considerable differences
in the structures of the protein monomers and their
complexes were not found (Jones and Thornton, 1996;
Muegge et al., 1998). However, most studies revealed
various structural changes occurring upon complex formation. These changes were denoted ‘induced-fit’ effects
(Betts and Sternberg, 1999; Decrescenzo et al., 2000;
Kimura et al., 2001; McCammon, 1998; Sundberg and
Mariuzza, 2000). Protein–protein interaction can induce
changes in the positions of the side chains of amino acids,
motion of the main chain (especially if it is a loop), or
domain (Betts and Sternberg, 1999; Carr et al., 1997; Davies
and Cohen, 1996; Jones and Thornton, 1996; Wall et al.,
1998). The analysis of the conformational changes in
lysozyme induced by binding of various antibodies showed
that some amino acids could deviate by up to 8 Å (Davies
and Cohen, 1996). It was shown that the rearrangement in
the protein backbone appeared to be due to low-energy
conformational changes, which enable H-bond formation
and packing of the amino acid residues (Janin, 2000). The
difference in data may be attributed to mechanisms
responsible for conformational changes during assemble
of permanent and non-obligate complexes. The former
operate during protein folding, which is accompanied by
mutual optimization of the interacting protein structures.
Proteins possessing prefolded structures form non-obligate
complexes. They have limited conformational freedom for
maximal optimization of the subunit structures. This results
in formation of cavities, the presence of water molecules at
the complex interfaces, non-optimal hydrogen bond geometry (Dall’Acqua et al., 1998; Vaughan et al., 1999; Xu et
al., 1997b) etc. The driving force for protein structure
adaptation is the decrease of free energy of the complexes.
Thermodynamics and kinetics of protein–protein
interactions
Thermodynamics gives the theoretical basis for understanding the processes of protein–protein interaction. The
formation of the protein–protein complex may be written as:
A‡B
ka
!AB
kd
Copyright # 2002 John Wiley & Sons, Ltd.
…1†
where kd is the first-order rate constant for the dissociation
reaction and ka is the second-order rate constant for the
association reaction. Their ratio is the equilibrium constant
for association (Ka) or for dissociation (Kd) according to the
law of mass action that is usually written as:
‰AŠ‰BŠ
1
kd
ˆ Kd ˆ
ˆ
‰ABŠ
Ka ka
…2†
although the use of the values of activity instead of reactant
concentrations is more correct.
The thermodynamic parameters have been determined by
numerous experimental methods: calorimetric (isothermal
titration and differential scanning calorimetry) methods
(Aoki et al., 1998a; Leavitt and Freire, 2001), UV–vis
absorption methods (Lehnerer et al., 1998), fluorescence
methods (Davydov et al., 1996), and analytical ultracentrifugation (Chirlando et al., 1995). Recently optical
biosensor methods have been introduced. These methods
are of two types, resonant mirror (Ivanov et al., 1999a,
2001) and surface plasmon resonance (Glaser and Hausdorf,
1996). These types allow recording of complex formation in
real time (without special labels) by recording the change of
refraction index of the medium during the complex
formation.
The interrelationship between the main thermodynamic
parameters characterizing complex formation, such as
Gibbs free energy (DG), enthalpy change (DH), entropy
change (DS), heat capacity difference (DCp) can be
described by the following equations:
G0 ˆ
RT ln Kd
G ˆ H
TS
Cp ˆ dH=dT ˆ T d…S†=dT
…3†
…4†
…5†
where T = temperature, DG0 = standard free energy change
and R = gas constant.
The free energy of protein–protein complex formation is
linked to the equilibrium constant or affinity by eqn (3), so it
is possible to estimate the DG0 value by determining the Kd
value. The Kd values for protein–protein complexes are
within the range 10 4–10 14 M which corresponds to DG
values of 6–19 kcal/mol (Janin, 2000).
The Gibbs free energy indicates the favourable direction
of processes. Changes of Gibbs energy are related to
changes of enthalpy (DH) and entropy (DS) [eqn (4)]. With
respect to protein–protein interaction, this equation reflects
two opposite tendencies, decrease of energy of the system
and complex dissociation due to Brownian and intramolecular vibration motions. Change of enthalpy depends on
hydrogen bond formation, electrostatic and van der Waals
interactions, whereas the change of entropy component
depends on changes of conformational freedom of the
system. Conformational entropy is often subdivided into
backbone and side chain contributions (Brady and Sharp,
1997). The backbone conformation entropy dominates in
protein folding, but it has a modest contribution to protein–
protein interactions when backbone changes are minor
(Stites, 1997). The main contribution of the conformation
entropy in the protein–protein interaction is usually the side
chain component (Brady and Sharp, 1997). The solvent and
association entropy are the other important components of
J. Mol. Recognit. 2002; 15: 405–422
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
entropy. Protein–protein complex formation leads to release
of water molecules from the surfaces of the protein interface
into the solvent; this generally results in an increase of
solvent entropy (Brady and Sharp, 1997). Protein complex
formation is accompanied by a reduction of the translational
and rotational freedom of partners that results in a change of
association entropy (Brady and Sharp, 1997). When the net
entropy change is positive, the protein–protein interaction is
entropy-driven; in the opposite case, the enthalpy is the
primary driving force of the interaction. Analysis of
enthalpy and entropy changes of protein complex formation
(69 complexes) demonstrates that, at physiological temperature, enthalpy favours protein–protein interaction in
74% of cases, while entropy favours formation in 55% of
the complexes (Stites, 1997). At different temperatures, the
leading driving force can be different. The interaction of hen
egg white lysozyme and Fab D 1.3 was driven by enthalpy
(at temperature below 23 °C), by enthalpy and entropy
(between 23 and 35 °C), and only by entropy (above 35 °C;
Zeden-Lutz et al., 1997). In most cases, the effects of
enthalpy and entropy are opposite. This leads to enthalpy/
entropy compensation that results in small changes in DG
values (Brady and Sharp, 1997).
The description of protein–protein binding requires
determination of the hydration states of hydrophobic groups
at interfaces that is reflected in a change DCp (Janin, 1995).
These groups become accessible to water molecules during
complex dissociation. The translocation of the hydrophobic
groups from water to non-polar environment is characterized by a large negative DCp value. Since in most protein–
protein complexes the DCp values are negative, this
indicates the vital importance of the hydrophobic interaction
in protein complex formation (Stites, 1997). For several
protein complexes a correlation between DCp and the values
of hydrophobic interaction was found (Gomez and Freire,
1995). We showed that in electron-transport monooxygenase systems containing cytochrome P450 the hydrophobic
force plays the major role in complex formation between
membrane bound cytochrome P4502B4 and its redox
partners cytochrome b5 and NADPH cytochrome P450
reductase. However it was shown that the electrostatic
interaction reduces the rate of formation of these complexes,
although it increases complex stability (Archakov and
Ivanov, 1999).
When proteins form tight complexes, kinetic measurement of Kd is preferable to equilibrium methods. The
equilibrium constant (Kd) represents the ratio of dissociation
(koff) and association (kon) rate constants. Typical association constants are in the range from 105 to 107 M 1 s 1
(Janin, 1995, 2000). The fastest complex formation rate was
found for the interaction of barnase with barstar
(2 109 M 1 s 1; Schreiber et al., 1997). The association
rate can reach nearly diffusion-limited values (Gloss and
Matthews, 1998; Janin, 1995). Typical Kd and koff values
vary from 10 6 to 10 14 M, and from 103 to 10 7 s 1,
respectively. For example, the lifetime of an antigen–
antibody complex may be about a year (koff < 10 6 s 1;
Yeung et al., 1995), whereas for protein complexes of the
cytochrome P450 electron-transport system this parameter
is limited to several minutes (Archakov and Ivanov, 1999;
Ivanov et al., 1999b, 2001). Usually point mutations at
interfaces reduce the affinity of the complex. As a rule a
Copyright # 2002 John Wiley & Sons, Ltd.
409
mutation results in increasing koff values and a minor effect
on kon values (Janin, 2000; Schreiber et al., 1997).
The relationship between equilibrium and rate constants
depends on the mechanism of protein–protein interaction.
The simplest mechanism is a one-step reaction, when
proteins form a complex without conformational adaptation
to each other [equation of reaction is the same as eqn (1)]. In
a two-step mechanism, when complex formation is
accompanied by conformational changes of monomers,
the reaction may be written as:
A‡B
0 k2
k1
! AB ! AB
k 2
1
…6†
k
where AB' is an intermediate complex before conformational changes. Then the Kd value is:
Kd ˆ
k 1k 2
ˆ K1 K2
k1 k2
…7†
Usually k2 is larger than k 2 and this shifts the reaction to
the right. When conformational changes are faster (in
comparison to intermediate complex dissociation, i.e. k2 is
large relative to k 1), eqn (6) reduces to eqn (1). A more
complex situation appears when electron transfer proteins
form protein–protein complexes. The minimal reaction
scheme for bimolecular complex formation of oxidized
electron acceptor and reduced electron donor with electron
transfer reaction consists of five steps: association of
proteins, equilibration of their energy levels, electron
transfer, relaxation of protein complex of monomers with
changed red-ox states and complex dissociation (Mathews
et al., 2000). So the fit between kinetic and equilibrium data
depends on a scheme that better represents the mechanism
of protein–protein interaction.
It is tempting to subdivide the process of protein–protein
interaction into two possible mechanisms responsible for
complex formation and stabilization. The first might underline the protein recognition followed by subsequent complex
stabilization due to direct docking of protein monomers. In
this case long-distance electrostatic forces determine the
oriented factor. However, at this stage the thermodynamic
barrier exists and the complex formation constant must be
below the diffusion-limited constant (kD). The second
mechanism represents only random collisions of the proteins
monomers (kon → kD) with subsequent fixation of the
complexes formed, which allows a high thermodynamic
barrier to be overcome. Complex formation is especially
favourable when kon → kD and koff → ?. In the case of
permanent complexes the situation is much more complex
because their formation appears to be a continuation of the
folding of three-dimensional structures and cannot be
evaluated by such simple thermodynamic considerations.
Recognition requires the directed forces of interaction
such as hydrogen bonds and electrostatic forces, whereas the
binding energy is probably also determined by hydrophobic
forces. Mutational analysis and analysis of the influence of
ionic strength on the interaction of the T lymphocyte cell–
cell recognition molecule CD2 with its ligand—CD48
showed little contribution of charged residues of the
contacted surface of CD2 to binding energy of their
interaction, whereas the loss of these charged residues leads
to marked reduction of ligand-binding specificity (Davis et
al., 1998). For the human growth hormone receptor
J. Mol. Recognit. 2002; 15: 405–422
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A. V. VESELOVSKY ET AL.
complex, it was shown that the hydrophobic area plays the
major role in the binding ability. Polar and charged residues
surrounding this area are less important for binding (Wells,
1996). Eight amino acids (six of them are hydrophobic) out
of 31 at the receptor interfaces of human growth hormone
account for about 85% of the binding energy (Wells, 1996).
The receptor site contains nine amino acids (six amino acids
are hydrophobic) that can account for all the binding affinity
(Clackson and Wells, 1995). Numerous directed-mutagenesis experiments demonstrate that in some cases mutation of
only one amino acid destroys protein–protein binding (Aoki
et al., 1998b; Behlke et al., 1998; Chen et al., 2000;
Eubanks et al., 2000; Gomez-Moreno et al., 1998; Grant et
al., 2000; Koltzscher and Gerke, 2000; Lin et al., 1999;
Lomax et al., 1998; Martin et al., 1999a; Ramadevi et al.,
1998; Saarela et al., 1998; Scott et al., 2000; Sengchanthalangsy et al., 1999; Sundberg et al., 2000; Thomas et al.,
1998; Zeng et al., 1999), whereas in other cases only a few
residues affect binding affinity (Clackson and Wells, 1995;
Jackson, 1999; Pons et al., 1999; Wells, 1996) or complex
stability (Martin et al., 1999b; Mateu and Fersht, 1998;
Vaughan et al., 1999; Xie et al., 1999). It is suggested that
most of the binding energy is related to only a few amino
acids from the interface, so-called ‘hot spots’ (Bogan and
Thorn, 1998; Bradshaw et al., 2000; Cunningham and
Wells, 1997; Hu et al., 2000b; Kuhlmann et al., 2000;
McInnes et al., 2000; von Kries et al., 2000).
Thus, the same forces are involved in protein–protein
interaction, protein folding and ligand–receptor interaction.
The dominant factor for permanent complex formation is
the hydrophobic force as for protein core folding. Thus such
complex formation has some similarities with folding
process. On the other hand, various forces participate in
formation of the non-obligate complexes and those properties of the contact surfaces are more complex.
Morphology of protein–protein interfaces
So far we considered the properties and the driving forces of
the protein–protein interaction without taking into consideration their spatial distributions, but examination of their
average values cannot give the correct notion of the protein–
protein interactions. The distribution of these properties is
not chaotic and corresponds to their functional properties.
The permanent complexes have hydrophobic surfaces at
their interfaces, which differ insignificantly from the protein
core (Tsai et al., 1997b). The morphology of the interfaces of
the non-obligate complexes is more variable (Larsen et al.,
1998) (Plate 1). Analysis of the morphology of protein–
protein interfaces showed that one-third of protein complexes have interfaces with a well-defined hydrophobic core
surrounded by a ring of polar groups [Larsen et al., 1998;
Plate 1(A)]. The water molecules are usually located in these
rings (Davies and Cohen, 1996; Larsen et al., 1998). Such
interfaces were shown in several studies (Tochio et al., 1999;
Wells, 1996). The other two-thirds of protein interfaces have
mixed hydrophilic properties without a definite continuous
hydrophobic patch [Larsen et al., 1998; plate 1(B)]. This
type of protein interface has mixed short hydrophobic
patches, polar groups and intersubunit hydrogen bonds
(Larsen et al., 1998). Water molecules are located at the
Copyright # 2002 John Wiley & Sons, Ltd.
protein interfaces, usually in cavities (Davies and Cohen,
1996; Larsen et al., 1998; Xu et al., 1997b). Monomers
forming non-obligate complexes are either in polar solution
or in the bound state in the complex. In the latter case they
interact with each other, and their contact areas are shielded
from the environment. In this case the hydrophobic surfaces
are more optimal. At the same time, in the polar solution they
should be hydrophilic enough to avoid non-specific aggregation, and whenever possible to shield the hydrophobic area of
contact surface from the solvent. This is achieved by
arrangement of the charged and polar groups around the
hydrophobic area or by decreasing the large continuous
hydrophobic patches by ‘dissemination’ of polar groups.
CHANGES OF PROTEIN–PROTEIN
INTERACTIONS
Since protein–protein interactions play a critical role in
living systems, they are controlled by numerous intracellular mechanisms and physico-chemical factors.
Temperature, ionic strength, pH and other physicochemical factors can influence protein–protein interactions.
At high temperature, head shock protein 90 oligomerizes
and shows a new chaperone activity (Yonehara et al., 1996).
The ionic strength of solution can affect the oligomeric
states of protein (Brazil et al., 1998; Shima et al., 1998).
Ionic strength influences the kinetics of protein–protein
interaction. For example, a decrease of ionic strength by one
order of magnitude leads to the reduction to the same degree
of kon and koff values of interaction of partners of
cytochrome P450 monooxygenase systems (Archakov and
Ivanov, 1999). This suggests the existence of a thermodynamic barrier both of the stage of complex formation and
its dissociation. The stability of protein complexes also
depends on pH (Gibas et al., 1997; Xie et al., 1998, 1999).
The mechanisms responsible for these changes include
charge masking (Gibas et al., 1997), conformational
changes leading to hydrophobic surface exposure (Valentemesquita et al., 1998), and changes of association/dissociation rate equilibrium (Xie et al., 1998). Chelators (such as
EDTA) can induce disintegration of a metal-containing
complex in which metal ions are involved in complex
formation (e.g. insulin; Murray-Rust et al., 1992).
Some proteins form dimers via disulphide bridges. This
process may occur spontaneously (Bell et al., 1998; Le et
al., 2000; Pace et al., 1999) or during termoinactivation
(Sasvari and Asboth, 1998) and free radical action (Kato et
al., 2000). This process can be regulated by chaperones
(Bass et al., 1998) and protein disulphide isomerases
(Markus and Benezra, 1999).
Cells can regulate the oligomerization state of their
proteins by covalent modification. The latter includes
phosphorylation (Behlke et al., 1998; Dare et al., 1999;
Eisenmessers and Post, 1998), glycosylation (Bell et al.,
1998; Tsuda et al., 2000), sulfation (Kehoe and Bertozzi,
2000), palmitoylation (Dunphy and Linder, 1998) and
myristoylation (Taniguchi, 1999). A well-known example
of regulation of the protein–protein interaction by covalent
modification is phosphorylation in the signal transduction
cascade (Eyster, 1998).
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PROTEIN–PROTEIN INTERACTIONS AND DRUGS
Plate 1. Examples of morphology of protein±protein interface. (A) Surface with continuous hydrophobic patch (Bence±Jones
immunoglobulin; PDB1REI); (B) surface without continuous hydrophobic patch (endonuclease; PDB1SMN). Hydrophobicity
increases in the following order: blue±green±brown.
Copyright # 2002 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2002; 15
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
411
Figure 2. Structures of dimerizers, their anchors and linkers.
Another mechanism influencing protein–protein interaction is noncovalent ligand binding. The latter can induce
formation (Laitinen et al., 2001; Ratovitski et al., 1999;
Wells, 1996) or stabilization of protein complexes (Okada,
1998; Rafferty et al., 1999; Smyczynski et al., 2000), and
also their dissociation (Rodriguezcrespo et al., 1998; Saito
et al., 1998) or prevention of another complex formation
(Garnier et al., 1998). Ligand binding can also lead to
enzyme inhibition (Huang et al., 1997; Ju et al., 1998;
Kojima et al., 1998). The most studied processes of the
regulation of the protein–protein interaction by noncovalent ligand binding are receptor systems responsible
for cell signalling (Klemm et al., 1998). For example,
human growth hormone (hGH) induces dimerization of its
receptor. At the first stage, hGH interacts with the
monomeric form of its receptor. During the next stage, the
second subunit of the receptor interacts with this complex
and the resultant trimer complex becomes active and
capable of transducing a signal (Wells, 1996). Such a
mechanism explains the difference between the effect of
agonists and antagonists. An agonist does induce a change
in the conformation of receptor subunit leading to the
receptor dimerization, whereas an antagonist does not
(Abdalla et al., 1999; Wells, 1996).
Thus, protein–protein interactions play an important role
Copyright # 2002 John Wiley & Sons, Ltd.
in cells and they are regulated by different mechanisms.
Therefore, artificial induction or disintegration of protein
complexes can lead to different physiological responses of
the cell. Over several years, different research groups have
tried to design compounds which could directly influence
protein–protein interactions. At the present moment, several
compounds inducing protein dimerization (dimerizers;
Austin et al., 1994; Clemons, 1999; Michnick, 2000), and
compounds preventing dimer formation (inhibitors of
dimerization, ID; Cochran, 2001; Way, 2000; Zutshi et
al., 1998), have been discovered or designed.
ARTIFICIAL SYSTEMS
Dimerizers
Many cell-signalling pathways are initiated by protein–
protein dimerization. So the main idea of dimerizer design
consists in induction of such dimerization by small
molecules and activation of a certain cell signal transduction
pathway. Since dimerizers must interact with two separate
proteins, they consist of three parts: two anchor groups
interacting with the proteins and a long linker between them
(Fig. 2). The chemical structure of each anchor group can be
J. Mol. Recognit. 2002; 15: 405–422
412
A. V. VESELOVSKY ET AL.
either identical (Amara et al., 1997; Blau et al., 1997;
Keenan et al., 1998) or different (Belshaw et al., 1996). At
present the anchor groups employed are FK506, rapamycin,
cyclosporin, coumermycin and others (Austin et al., 1994;
Belshaw et al., 1996; Clemons, 1999; Ho et al., 1996;
Kopytek et al., 2000; Smith and Vanetten, 2001). Linkers
consist of 5–16 atoms (Amara et al., 1997; Keenan et al.,
1998). FK1012 is the most frequently used dimerizer. It is a
non-toxic lipid-soluble dimer of the drug FK506. The latter
selectively interacts with endogenous FK506-binding protein 12 (FKBP12) and this complex interacts with the
cellular phosphatase, calcineurin, and inhibits it (Clemons,
1999). The concentrations required for induction of
dimerization by such compounds vary within a relatively
narrow range of 1–10 nM (Amara et al., 1997; MacCorkle et
al., 1998). The effectiveness of dimerizers depends on the
anchors affinity (Keenan et al., 1998) and the length of the
linkers (Amara et al., 1997; Keenan et al., 1998). There
were several attempts to design simple-analogues of FK506
(Clackson et al., 1998; Keenan et al., 1998). However, they
were much less effective (Keenan et al., 1998).
At present dimerizers are used only in laboratory practice
to study cell proliferation (Blau et al., 1997), transcription
(Amara et al., 1997; Belshaw et al., 1996), apoptosis
(Amara et al., 1997; MacCorkle et al., 1998). However,
these systems can be potentially used for clinical purposes,
particularly in gene therapy. Dimerizers can be employed
for induction of cell proliferation (Blau et al., 1997), or for
elimination of transferred gene product and genetically
modified cells by dimerizer-inducing apoptosis (Amara et
al., 1997; MacCorkle et al., 1998). It is suggested to insert
the additional chimeric gene, coding apoptosis-inducing
protein with dimerizer-interacting domain, in genetically
modified cells. When it is necessary to eliminate such
modified cells, the dimerizers could be introduced into the
cells. Since the dimerizer interacts with its target inducing
apoptosis in the modified cells only, this will cause death of
modified cells only (MacCorkle et al., 1998).
Inhibitors of dimerization
Numerous proteins act either as oligomer complexes, or
they change aggregate states during their action. So
inhibitors of dimerization (IDs) may prevent formation of
an active dimer. Such inhibitors have been discovered for
three HIV enzymes (protease, reversed transcriptase,
invertase; Bouras et al., 1999; Morris et al., 1999; Sourgen
et al., 1996; Zutshi et al., 1998), ribonucleotide reductase
(Liuzzi et al., 1994) and DNA polymerase (Digard et al.,
1995) of herpes simplex virus, human gluthatione reductase
(Nordhoff et al., 1997), phosphatidylinisitol 3-kinase (Eaton
et al., 1998), virus capsid (Hilpert et al., 1999; Prevelige,
1998) and some others (Beaulieu et al., 1999; Brickner and
Chmielewski, 1998; Chen et al., 2001; Crump et al., 1998;
Findeis, 2000; Gay et al., 1999b; Ghosh et al., 1999; Hart et
al., 1999; Kim et al., 1999; Li et al., 1998; Lou et al., 1999;
Pacofsky et al., 1998; Prasanna et al., 1998; Saito et al.,
1998; Singh et al., 2001; Vu et al., 1999; Usui et al., 1998;
Yao et al., 1998, 1999). Most of the discovered IDs are
peptides resembling dimer interfaces (Zutshi et al., 1998),
although peptidomimetic molecules or small organic molCopyright # 2002 John Wiley & Sons, Ltd.
ecules have also been found (Bouras et al., 1999; Findeis,
2000; Gay et al., 1999b; Sennequier et al., 1999; Souroujon
and Mochly-Rosen, 1998; Yao et al., 1999; Zutshi et al.,
1998).
The activity of such inhibitors (expressed as IC50 or Ki
values) varied from low nanomolar (Eaton et al., 1998;
Schramm et al., 1999; Shultz and Chmielewski, 1999) to
micromolar concentration range (Beaulieu et al., 1999;
Sennequier et al., 1999; Usui et al., 1998; Yao et al., 1999;
Zutshi et al., 1998). Peptide inhibitors of protein dimerization exhibit two important features. The longer peptides
representing dimer interfaces are more potent inhibitors
than their shorter derivatives (Digard et al., 1995; Ghosh
and Chmielewski, 1998; Vu et al., 1999). Another important
feature of peptide IDs is a possibility of improvement of
their inhibitory activity by optimizing their amino acid
composition and sequence. Using site-directed mutagenesis
or methods of combinatorial chemistry more potent peptide
inhibitors (than the primary peptide fragments from the
dimer interface) have been developed (Hart et al., 1999; Li
et al., 1998; Morris et al., 1999; Pacofsky et al., 1998;
Shultz and Chmielewski, 1999; Zutshi et al., 1998).
HIV protease is the most studied protein target for IDs
(Ast et al., 1998; Bouras et al., 1999; Schramm et al., 1999;
Shultz et al., 2000; Shultz and Chmielewski, 1997, 1999;
Ulysse and Chmielewski, 1998; Zutshi et al., 1997, 1998).
The active dimer of HIV protease is formed by interaction
of two b-sheets from each subunit (Wlodawer, Vondrasek,
1998). It was initially found that peptides corresponding to
the N- and C-termini of HIV protease inhibit its activity
(Zutshi et al., 1997). Subsequently it was shown that some
synthetic peptides are more potent inhibitors of HIV
protease than those derived from the subunit interfaces
(Ulysse and Chmielewski, 1998; Zutshi et al., 1997, 1998).
The first generation of such inhibitors had flexible linkers
(Shultz and Chmielewski, 1997; Ulysse and Chmielewski,
1998; Zutshi et al., 1997, 1998) with optimal distance
between peptides fragments of about 10 Å (Zutshi et al.,
1998). The most potent inhibitor containing a flexible linker
had an IC50 of 25 nM (Zutshi et al., 1997). Inhibitors with
rigid linkers (‘molecular tongs’), containing tri- or tetrapeptidic arms attached to pyridinediol or naphthalenendiol
were less active (Ki about 0.56–4.5 mM) (Bouras et al.,
1999). High inhibitory potency (Ki in a low nanomolar
range) was shown for lipopeptides containing peptide,
linker and lipid (Schramm et al., 1999). Recently a nonpeptidic inhibitor of HIV-1 protease dimerization was
designed (Song et al., 2001).
In addition to ability to prevent protein dimerization,
some IDs can also cause dimer dissociation. It was found
that the imidazole derivative, clotrimazole, induced dissociation of inducible NO synthase into subunits in the
absence of L-arginine and tertahydrobiopterin, whereas
other derivatives only prevented dimerization (Sennequier
et al., 1999). The fungal metabolite, tryprostatin A, induced
reversible disruption of the cytoplasmic microtubule
assembly in 3Y1cells (Usui et al., 1998). Thiol reagent
4,4'-dithiodipyridine, which covalently binds to cystein-458
of GroEL chaperonin, induced its dissociation (Bochkareva
et al., 1999). The attacked cysteine is located in an almost
inaccessible region between subunits of the protein and the
ability of large a SH-reagent to interact with its target
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PROTEIN–PROTEIN INTERACTIONS AND DRUGS
suggests that during molecular dynamics this protein turns
into an opened state. Peptides, which induce dissociation
into monomers of HIV-1 protease and integrase, were found
(Maroun et al., 2001; Park and Raines, 2000).
Inhibition of protein(peptide)–receptor interactions
Protein–protein interactions play the key role in receptormediated signal transduction. This process can be subdivided into several steps: interaction of the protein(peptide)
hormones with their receptors followed by interaction of the
hormone–receptor complex with other proteins of the signal
transduction cascades. Modification of each of these
interactions can be employed to change the cell metabolism.
Design of inhibitors preventing receptor-mediated signal
transduction may be similar to that of inhibitors of HIV
protease dimer formation (see above). The design of
interleukin-1 receptor antagonists was initiated by discovery
of small (10–12 residues in length) peptides exhibiting
modest inhibitory activity (IC50 about 10 5 M) (Yanofsky et
al., 1996). The subsequent screening of a peptide library
resulted in the discovery of 15 residue peptides with IC50 of
about 2 nM. The substitution of proline for azetidine, aminoterminal acetylation and carboxy-terminal amidation gave
peptidomimetic with IC50 about 0.5 nM (Cunningham and
Wells, 1997). Other ways for the design of antagonists
employed combinatorial chemistry, high-throughput
screening and computer simulation methods. These
methods were used for design of receptor antagonists for
vascular endothelial growth factor (Aviezer et al., 2000),
somatostatin (Rohrer et al., 1998), neuropeptide Y (Parker
and Parker, 2000), thromboxane A2 (Marusawa et al.,
1999), protease-activated receptors (Andrade-Gordon et al.,
1999; Fujita et al., 1999; Hoekstra et al., 1998) and others
(Beebe et al., 2000; Chackalamannil et al., 2001; Freidinger, 1999; Nicole et al., 2000; Yudt and Koide, 2001).
There are many examples of successful design of
effective nonpeptide ligands for different types of receptors
of peptide(protein) hormones (Freidinger, 1999). On the
basis of these results, peptidomimetics for these receptors
were subdivided into three types (Ripka and Rich, 1998):
peptidomimetics of the first type simulate the peptide
backbone; peptidomimetics of the second type are small
molecules that elicit agonist or antagonist activity, but they
do not mimic the structure of native ligands; non-peptide
molecules of the third type mimic the main binding
elements of the peptide ligands.
The ability to regulate signal transduction at the various
steps by the inhibition of protein–protein interactions was
shown using a signal cascade induced by light adsorption by
rhodopsin. Retinal light absorption results in conformational change in the rhodopsin molecule and its interaction
with the membrane bound G protein (Gt). The latter
interacts with cGMP phosphodiesterase. Interaction of
phosphorylated rhodopsin with arrestin (this protein blocks
the interaction of rhodopsin with Gt) interrupts the signal
transduction. Different peptides that can stop all stages of
cascade induced by the light adsorption by rhodopsin were
identified (Zutshi et al., 1998).
Recently it was shown that many receptors coupled to Gprotein act as oligomer complexes (Overton and Blumer,
Copyright # 2002 John Wiley & Sons, Ltd.
413
2000; Salahpour et al., 2000) and peptides, the structural
analogues of the receptor transmembrane domains, can
destroy their interaction (Tarasova et al., 1999).
Numerous investigations have been done in an attempt to
design IDs for proteins possessing stable domains, which
interact with receptors: SH2 domain (Alligood et al., 1998;
Beaulieu et al., 1999; Burke et al., 1999, 2001; Cody et al.,
2000; Davidson and Martin, 2000; Eaton et al., 1998; Fretz
et al., 2000; Furet et al., 1999, 2000; Gao et al., 2000; Gay et
al., 1999a,b; Garciaecheverria, 2001; Hart et al., 1999; Kim
et al., 1999; Lou et al., 1999; Metcalf et al., 2000; Niimi et
al., 2001; Pacofsky et al., 1998; Rickles et al., 1998;
Schoepfer et al., 1998, 1999; Shakespeare, 2001; Shakespeare et al., 2000; Violette et al., 2000; Vu, 2000; Vu et al.,
1999; Walker et al., 2000; Yao et al., 1999), SH3 domain
(Dalgarno et al., 1997; Nguyen et al., 2000; Witter et al.,
1998) and PDZ domain (Fuh et al., 2000).
FUTURE PERSPECTIVES
Protein–protein interactions play a pivotal role in numerous
cellular processes. The formation of permanent protein
complexes can be considered as the extended folding of
these proteins. Subunits of such proteins have similar
structure, amino acid distribution over dimer interfaces and
forces participating in the protein interaction as in the
protein core (Srivastava and Sauer, 2000; Stites, 1997;
Tamura and Privalov, 1997; Tsai et al., 1996). Final folding
of subunits occurs during formation of such complexes
(Srivastava and Sauer, 2000; Tsai et al., 1997b; Wallace and
Dirr, 1999). However, the structure and properties of the
protein interfaces of the non-obligate complexes have a dual
nature. They share similarity with the protein core and
resemble enzyme active site surfaces. High specificity of the
interaction of these proteins suggest complementarity of
dimer subunits and therefore distribution of their unique
properties over protein interfaces. In this sense, they can be
compared with active sites of enzymes, which are also
influenced by their substrates. For example, protein–protein
interaction and binding of peptide inhibitors to the active
site of protease represent the same process (Wlodawer and
Vondrasek, 1998). So the protein interfaces of the subunits
of the non-obligate complexes can be considered to some
extent as the equivalent of enzyme active sites. They
represent protein regions for which new biologically active
compounds, in particular drugs, can be designed.
Numerous results during recent years unquestionably
support validity of such an approach. Successful examples
have been found using different cells and enzymes. The
designed compounds can act as dimerizers (Austin et al.,
1994; Clemons, 1999), and also as agents preventing this
process (inhibitors of dimerization and antagonists of
peptide/protein receptors; Beeley, 2000; Cochran, 2000,
2001; Cody et al., 2000; Freidinger, 1999; Way, 2000;
Zutshi et al., 1998). Many designed compounds possess
high activity and selectivity.
Dimerizers are also used to change cell functions. This
may be achieved by acting at various systems. Therefore at
present their employment in clinical practice is limited by
our insufficient knowledge about these processes. Since
dimerizers consist of three parts, they are rather large
J. Mol. Recognit. 2002; 15: 405–422
414
A. V. VESELOVSKY ET AL.
molecules, and their transport to a particular destination
through numerous cellular and tissue barriers may represent
a serious problem. At the same time, the universality of such
systems may have important advantages. The same
dimerizer can be used for many purposes since its action
depends on the constructed target (original protein with
fused dimerizer binding domain). The further development
of this field will probably be focused on detailed
characterization of the cell systems, which can be modified
by dimerizers, and in designing new dimerizers (the binding
parts and linkers) for increasing selectivity for existing
domain(s) or discovering new ones, in particular, for design,
the anchor part for mutant binding domain to exclude a
capability of dimerizer binding to normal cell proteins
containing such a domain (Clackson et al., 1998; Yang et
al., 2000).
Protein–protein interactions have big potential as a new
class of targets for the novel generation of drugs. However,
the search for new potential targets for these drugs requires
some common criteria. In our viewpoint, these criteria must
include the following points:
(1) Low-molecular-weight compounds are more effective
when the sizes of contacted interfaces are quite small. In
this case we may expect that the binding energy of
proteins is not high, and the small molecule can interact
with a considerable part of these contact surfaces.
(2) IDs should preferably interact with amino acids representing ‘hot spots’ of the interface. In this case the
interaction of IDs with such amino acids would
effectively prevent protein complex formation. The
inhibitors of dimerization can be applied both for
modification of regulator processes (as dimerizers),
and for prevention of formation of the enzyme active
form.
Methods for IDs design can be the same as for inhibitors
binding at the active site of an enzyme. These include
experimental methods [combinatorial chemistry (Beebe et
al., 2000; Hart et al., 1999; Nefzy et al., 1998), highthroughput screening (Lebl, 1999)], computer approaches
[molecular database mining (Loughney et al., 1999;
Marrone et al., 1997), computer-aided design (Fretz et al.,
2000; Marrone et al., 1997; Schoepfer et al., 1998; Zeng,
2000)] and others. The classic pathway for drug design
(peptide–peptidomimetic–small organic molecule) is also
applicable (Beeley, 2000; Liu, 1999; Stigers et al., 1999;
Vu, 2000) and several positive results have recently been
reported (Alligood et al., 1998; Beaulieu et al., 1999;
Cunningham and Wells, 1997; Freidinger, 1999; Hart et al.,
1999; Schoepfer et al., 1998; Witter et al., 1998).
Recently, a new approach for the first step of ID design
has been proposed. It requires the development of a single
chain antibody against one interaction surface of one protein
and use of this antibody as a template for design of
inhibitors (Chrunyk et al., 2000).
Another important question in IDs design is the
possibility of obtaining relatively small compounds, since
peptides are not optimal for medicinal practice. ‘Unlike the
interactions of enzymes with non-protein substrates,
protein–protein interactions usually do not occur in tightly
binding pockets. Instead, interactions between proteins
Copyright # 2002 John Wiley & Sons, Ltd.
usually occur across large, flat surfaces’ (Way, 2000);
however, ‘most studies show that relatively few residues
within these large contact surfaces actually drive binding’
(Cunningham and Wells, 1997). Furthermore, it was noted
that in several studies a mutation of one amino acid leads to
full loss of protein dimerization ability or to significant
decrease of binding constants (Behlke et al., 1998; Eubanks
et al., 2000; Imai et al., 2000; Koltzscher and Gerke, 2000;
Lin et al., 1999; Lomax et al., 1998; Martin et al., 1999a;
Omata et al., 2000; Ramadevi et al., 1998; Saarela et al.,
1998; Sengchanthalangsy et al., 1999; Stenberg et al., 2000;
Thomas et al., 1998; Zeng et al., 1999). Also oxidation of
two methionine residues to methionine sulphoxide at the
dimer interface of HIV-2 protease resulted in inactivation of
this enzyme (Davis et al., 2000). There are some indications
that the IDs, interacting with such critical amino acids, may
be relatively small molecules. Nevertheless, the problem for
small IDs still exists.
Recently, a new strategy to design IDs to increase the
effectivity of such inhibitors has been proposed. At the first
step, ID interacts with its target non-covalently. This
interaction brings together weakly reactive groups of the
drug and amino acid side chain. At the second step, such
contacted groups covalently interact with each other (Way,
2000). The same strategy was used to design inhibitors of
dimerization of HIV protease and SH2 domain (Violette et
al., 2000; Zutshi and Chmielewski, 2000). The designed
molecules form disulphide bridges between drugs and
cysteine located at protein interface surfaces.
It is possible to assume that IDs can be a useful class of
compounds for design of antibiotic, antiviral and parasitic
drugs. There are two favourable features of IDs for such
drugs. Design of potential antibiotic interacting with the
active site of enzyme can be limited by the high structural
conservation of active sites, which can prevent an effective
distinction between the human and the pathogen enzymes.
The greater structural variability of protein–protein interfaces may supply a target for the effective differentiation
between the host and pathogen enzymes (Singh et al., 2001).
The second favourable feature of IDs is related to the
problem of overcoming antibiotic resistance of pathogens.
One of the preferential mechanisms of antibiotic resistance
is the mutation of amino acid in the active site of the
antibiotic-target enzyme. Since the substrate and drugs can
interact with the different groups at the active site, the
mutation of active site amino acid (but not catalytic
residues) can lead to decreased affinity of the drug with
little effect on substrate binding and enzyme activity.
Numerous data exist that a single mutation in one subunit of
protein–protein interface can destroy the protein–protein
interaction (see above). The conservation of protein
complex in this case requires the complementary mutation
in the second subunit. The simultaneous double mutation in
different subunits is much more unlikely, so it seems that the
essential amino acids of protein–protein interface are quite
conservative. It is also unlikely that pathogens acquire
resistance for IDs binding to such amino acid residues.
Recently, several compounds were found that may act as
inhibitors of dimerization and as dimerizers. It was shown
that some pyrrolidine derivatives are competitive inhibitors
of serum amyloid P component (SAP) glycoprotein binding
to amyloid fibrils. These low-weight compounds are also
J. Mol. Recognit. 2002; 15: 405–422
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
able to dimerize SAP molecules, leading to their rapid
elimination by the liver (Pepys et al., 2002).
Finally, one more speculation can be made. Since small
molecules can prevent the protein–protein interaction, it
might be possible to design (or discover) small compounds
that selectively inhibit protein folding. At present, no
experimental data exist, but it may become an interesting
field in drug design in the recent future.
All of this gives hopes that in the future the compounds
regulating protein–protein interactions will take a respect-
415
able place adjacent to ‘classic’ drugs that interact with
active sites of enzymes.
Acknowledgements
This work was partially supported by the Russian Foundation for Basic
Research (grants 01-04-48128, 99-04-48081) and a cooperation with
‘Janssen Pharmaceutica NY. Center for molecular design’. The authors
thank Dr A. E. Medvedev for valuable and helpful discussions.
REFERENCES
Abdalla S, Zaki E, Lother H, Quitterer U. 1999. Involvement of the
amino terminus of the B-2 receptor in agonist-induced
receptor dimerization. J. Biol. Chem. 274: 26079±26084.
Ahmad KF, Engel CK, Prive GG. 1998. Crystal structure of the BTB
domain from PLZF. Proc. Natl Acad. Sci. USA 95: 12123±
12128.
Alattia JR, Kurokawa H, Ikura M. 1999. Structural view of adherinmediated cell-cell adhesion. Cell Mol. Life Sci. 55: 359±367.
Alligood KJ, Charifson PS, Crosby R, Consler TG, Feldman PL,
Gampe RT Jr., Gilmer TM, Jordan SR, Milstead MW, Mohr C,
Peel MR, Rocque W, Rodriguez M, Rusnak DW, Shewchuk
LM, Sternbach DD. 1998. The formation of a covalent
complex between a dipeptide ligand and the src SH2 domain.
Bioorg. Med. Chem. Lett. 8: 1189±1194.
Amara JF, Clackson T, Rivera VM, Guo T, Keenan T, Natesan S,
Pollock R, Yang W, Courage NL, Holt DA, Gilman M. 1997. A
versatile synthetic dimerizer for the regulation of protein±
protein interactions. Proc. Natl Acad. Sci. USA 94: 10618±
10623.
Andrade-Gordon P, Maryanoff BE, Derian CK, Zhang HC, Addo
MF, Darrow AL, Eckardt AJ, Hoekstra WJ, McComsey DF,
Oksenberg D, Reynolds EE, Santulli RJ, Scarborough RM,
Smith CE, White KB. 1999. Design, synthesis, and biological
characterization of a peptide-mimetic antagonist for a
tethered-ligand receptor. Proc. Natl Acad. Sci. USA 96:
12257±12262.
Aoki M, Ishimori K, Fukada H, Takahashi K, Morishima I. 1998a.
Isothermal titration calorimetric studies on the asociations of
putidaredoxin to NADH-puridaredoxin reductase and
P450cam. Biochim. Biophys. Acta 1384: 180±188.
Aoki M, Ishimori K, Morishima I. 1998b. Roles of negatively
charged surface residues of putidaredoxin in interactions
with redox partners in P450cam monooxygenase system.
Biochim. Biophys. Acta 1386: 157±167.
Appling DR. 1999. Genetic approaches to the study of protein±
protein interactions. Methods 19: 338±349.
Archakov AI, Ivanov YuD. 1999. The optical biosensor study of the
protein±protein interactions with cytochrome P450s. In
Biophysics of Electron Transfer and Molecular Bioelectronics. Plenum: New York; 173±194.
Ast O, Jentsch KD, Schramm HJ, Hunsmann G, Luke W, Petry H.
1998. A rapid and sensitive bacterial assay to determine the
inhibitory effect of `interface' peptides on HIV-1 protease
coexpressed in Escherichia coli. J. Virol. Meth. 71: 77±85.
Austin DJ, Crabtree GR, Schreiber SL. 1994. Proximity versus
allostery: the role of regulated protein dimerization in
biology. Chem. Biol. 1: 131±136.
Aviezer D, Cotton S, David M, Segev A, Khaselev N, Galili N,
Gross Z, Yayon A. 2000. Porphyrin analogues as novel
antagonists of ®broblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis.
Cancer Res. 60: 2973±2980.
Banci L, Benedetto M, Bertini I, Delconte R, Piccioli M, Viezzoli
MS. 1998. Solution structure of reduced monomeric Q133M2
copper, zinc superoxide dismutase (SOD). Why is SOD a
dimeric enzyme? Biochemistry 37: 11780±11791.
Copyright # 2002 John Wiley & Sons, Ltd.
Bass J, Chiu G, Argon Y, Steiner DF. 1998. Folding of insulin
receptor monomers is facilitated by the molecular chaperones calnexin and calreticulin and impaired by rapid
dimerization. J. Cell Biol. 141: 637±646.
Beaulieu PL, Cameron DR, Ferland JM, Gauthier J, Ghiro E,
Gillard J, Gorys V, Poirier M, Rancourt J, Wernic D, LlinasBrunet M, Betageri R, Cardozo M, Hickey ER, Ingraham R,
Jakes S, Kabcenell A, Kirrane T, Lukas S, Patel U, Proudfoot J,
Sharma R, Tong L, Moss N. 1999. Ligands for the tyrosine
kinase p561ck SH2 domain: discovery of potent dipeptide
derivatives monocharged, nonhydrolyzable phosphate replacements. J. Med. Chem. 42: 1757±1766.
Beebe KD, Wang P, Arabaci G, Pei DH. 2000. Determination of the
binding speci®city of the SH2 domains of protein tyrosine
phosphatase SHP-1 through the screening of a combinatorial
phosphotyrosyl peptide library. Biochemistry 39: 13251±
13260.
Beeckmans S. 1999. Chromatographic methods to study protein±
protein interactions. Methods 19: 278±305.
Beeley NRA. 2000. Can peptides be mimicked? Drug Discov.
Today 5: 354±363.
Behlke J, Heidrich K, Naumann M, Muller EC, Otto A, Reuter R,
Kriegel T. 1998. Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14. Biochemistry 37: 11989±11995.
Bell SL, Khatri IA, Xu GQ, Forstner JF. 1998. Evidence that a
peptide corresponding to the rat Muc2 C-terminus undergoes
disulphide-mediated dimerization. Eur. J. Biochem. 253: 123±
131.
Belshaw PJ, Ho SN, Crabtree GR, Schreiber SL. 1996. Controlling
protein association and subcellular localization with a
synthetic ligand that induces heterodimerization of proteins.
Proc. Natl Acad. Sci. USA 93: 4604±4607.
Betts MJ, Sternberg MJE. 1999. An analysis of conformational
changes on protein±protein association: implications for
predictive docking. Protein Engng. 12: 271±283.
Bhattacharya A, Bhattacharyya B, Roy S. 1996. Fluorescence
energy transfer measurement of distances between ligand
binding sites of tubulin and its implication for protein±protein
interaction. Protein Sci. 5: 2029±2036.
Blatch GL, Lassle M. 1999. The tetratricopeptide repeat: a
structural motif mediating protein±protein interactions.
Bioessays 21: 932±939.
Blau AC, Petrson KR, Drachman JG, Spencer DM. 1997. A
proliferation swich for genetically modi®ed cells. Proc. Natl
Acad. Sci. USA 94: 3076±3081.
Bochkareva E, Safro M, Girshovich A. 1999. Interaction of 4,4'dithiodipyridine with Cys(458) triggers disassembly of
GroEL. J. Biol. Chem. 274: 20756±20758.
Bogan AA, Thorn KS. 1998. Anatomy of hot spots in protein
interfaces. J. Mol. Biol. 280: 1±9.
Bouras A, Boggetto N, Benatalah Z, Derosny E, Sicsic S,
Reboudravaux M. 1999. Design, synthesis, and evaluation
of conformationally constrained tongs, new inhibitors of HIV1 protease dimerization. J. Med. Chem. 42: 957±962.
Bradshaw JM, Mitaxov V, Waksman G. 2000. Mutational
investigation of the speci®city determining region of the
J. Mol. Recognit. 2002; 15: 405–422
416
A. V. VESELOVSKY ET AL.
Src SH2 domain. J. Mol. Biol. 299: 521±535.
Brady GP, Sharp KA. 1997. Entropy in protein folding and in
protein±protein interactions. Curr. Opin. Struct. Biol. 7: 215±
221.
Brazil BT, Ybarra J, Horowitz PM. 1998. Divalent cations can
induce the exposure of GroEL hydrophobic surfaces and
strengthen GroEL hydrophobic binding interactionÐnovel
effects of Zn2‡ GroEL interactions. J. Biol. Chem. 273: 3257±
3263.
Brickner M, Chmielewski J. 1998. Inhibiting the dimeric restriction
endonuclease EcoRI using interfacial helical peptides. Chem.
Biol. 5: 339±343.
Burke TR Jr, Luo J, Yao Z-J, Gao Y, Zhao H. 1999. Monocarboxylic-based phosphotyrosyl mimetics in the design of
Grb2 SH2 domain inhibitors. Bioorg. Med. Chem. Lett. 9: 347±
352.
Burke TR, Yao ZJ, Gao Y, Wu JX, Zhu XF, Luo JH, Guo RB, Yang
DJ. 2001. N-terminal carboxyl and tetrazole-containing
amides as adjuvants to Grb2 SH2 domain ligand binding.
Bioorg. Med. Chem. 9: 1439±1445.
Camacho CJ, Weng ZP, Vajda S, Delisi C. 1999. Free energy
landscapes of encounter complexes in protein±protein
association. Biophys. J. 76: 1166±1178.
Camacho CJ, Kimura SR, Delisi C, Vajda S. 2000. Kinetics of
desolvatation-mediated protein±protein binding. Biophys. J.
78: 1094±1105.
Carr PA, Erickson HP, Palmer AG. 1997. Backbone dynamics of
homologous ®bronectin type III cell adhesion domain from
®bronectin and tenascin. Structure 5: 949±959.
Chackalamannil S, Doller D, Eagen K, Czarniecki M, Ahn HS,
Foster CJ, Boykow G. 2001. Potent, low molecular weight
thrombin receptor antagonists. Bioorg. Med. Chem. Lett. 11:
2851±2853.
Chen C, Zhu YF, Liu XJ, Lu ZX, Xie Q, Ling N. 2001. Discovery of a
series of nonpeptide small molecules that inhibit the binding
of insulin-like growth factor (IGF) to IGF-binding proteins. J.
Med. Chem. 44: 4001±4010.
Chen H, Shi M, Guo ZY, Tang YH, Qiao ZS, Liang ZH, Feng YM.
2000. Four new monomeric insulins obtained by alanine
scanning the dimer-forming surface of the insulin molecule.
Protein Engng. 13: 779±782.
Chirlando R, Keown MB, Mackay GA, Lewis MS, Unkeles JC,
Gould HJ. 1995. Stochiometry and thermodinamics of the
interaction between the Fc fragment of human IgG1 and its
low-af®nity receptor Fc gamma RIII. Biochemistry 34: 13320±
13327.
Chrunyk BA, Rosner MH, Cong Y, McColl AS, Otterness IG,
Daumy GO. 2000. Inhibiting protein±protein interactions: a
model for antagonist design. Biochemistry 39: 7092±7099.
Clackson T, Wells JA. 1995. A hot spot of binding energy in a
hormone-receptor interface. Science 267: 383±386.
Clackson T, Yang W, Rozamus LW, Hatada M, Amara JF, Rollins
CT, Stevenson LF, Magari SR, Wood SA, Courage NL, Lu XD,
Cerasoli F, Gilman M, Holt DA. 1998. Redesigning an FKBPligand interface to generate chemical dimirizers with novel
speci®city. Proc. Natl Acad. Sci. USA 95: 10437±10442.
Clemons PA. 1999. Design and discovery of protein dimerizers.
Curr. Opin. Chem. Biol. 3: 112±115.
Cochran AG. 2000. Antagonists of protein±protein interactions.
Chem. Biol. 7: R85±R94.
Cochran AG. 2001. Protein±protein interfaces: mimics and
inhibitors. Curr. Opin. Chem. Biol. 5: 654±659.
Cody WL, Lin ZW, Panek RL, Rose DW, Rubin JR. 2000. Progress
in the development of inhibitors of SH2 domains. Curr.
Pharm. Des. 6: 59±98.
Cohen FE, Prusiner SB. 1998. Pathologic conformations of prion
proteins. A. Rev. Biochem. 67: 793±819.
Crump CM, Williams JL, Stephens DJ, Banting G. 1998. Inhibition
of the interaction between tyrosine-based motifs and the
medium chain subunit of the AP-2 adaptor complex by
speci®c tyrphostins. J. Biol. Chem. 273: 28073±28077.
Cunha CA, Romao MJ, Sadeghi SJ, Valetti F, Gilardi G, Soares
CM. 1999. Effects of protein-protein interactions on electron
transfer: docking and electron transfer calculations for
complexes between ¯avodoxin and c-type cytochromes. J.
Copyright # 2002 John Wiley & Sons, Ltd.
Biol. Inorg. Chem. 4: 360±374.
Cunningham BC, Wells JA. 1997. Minimized proteins. Curr. Opin.
Struct. Biol. 7: 457±462.
Dalgarno DC, Bot®eld MC, Rickles RJ. 1997. SH3 domains and
drug design: ligands, structure, and biological function.
Biopolymers 43: 383±400.
Dall'Acqua W, Goldman ER, Lin W, Teng C, Tsuchiya D, Li H,
Ysern X, Braden BC, Li Y, Smith-Gill SJ, Mariuzza RA. 1998. A
mutational analysis of binding interactions in an antigen±
antibody protein±protein complex. Biochemistry 37: 7981±
7991.
Dare S, Schumacher J, Rousseau P, Fourment J, Ebel C, Kahn D.
1999. Phosphorylation-induced dimerization of the FixJ
receiver domain. Mol. Microbiol. 34: 504±511.
Davidson JP, Martin SF. 2000. Use 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide mimics in
SH2 antagonists. Tetrahedron Lett. 41: 9459±9464.
Davies DR, Cohen GH. 1996. Interactions of protein antigen with
antibodies. Proc. Natl Acad. Sci. USA 93: 7±12.
Davis DA, Newcomb FM, Moskovitz J, Wing®eld PT, Stahl SJ,
Kaufman J, Fales HM, Levine RL, Yarchoan R. 2000. HIV-2
protease is inactivated after oxidation at the dimer interface
and activity can be partly restored with methionine sulphoxide reductase. Biochem. J. 346: 305±311.
Davis SJ, Davies EA, Tucknott MG, Jones EY, van der Merwe PA.
1998. The role of charged residues mediating low af®nity
protein±protein recognition at the cell surface by CD2. Proc.
Natl Acad. Sci. USA 95: 5490±5494.
Davydov DR, Knyushko TV, Kanaeva IP, Koen YM, Samenkova
NF, Archkov AI, Hui Bon Hoa G. 1996. Interactions of
cytochrome P450 2B4 with NADPH-cytochrome P450 reductase studied by ¯uorescent probe. Biochimie 78: 734±743.
Dear TN, Hainzl T, Follo M, Nehls M, Wilmore H, Matena K,
Boehm T. 1997. Identi®cation of interaction partners for the
basic-helix±loop±helix protein E47. Oncogene 14: 891±898.
Decrescenzo G, Grothe S, Lortie R, Debanne MT, Oconnormccourt M. 2000. Real-time kinetic studies on the interaction of
transforming growth factor alpha with the epidermal growth
factor receptor extracellular domain reveal a conformational
change model. Biochemistry 39: 9466±9476.
Digard P, Williams KP, Hensley P, Brooks IS, Dahl CE, Coen DM.
1995. Speci®c inhibition of herpes simplex virus DNA
polymerase by helical peptides corresponding to the subunit
interface. Proc. Natl Acad. Sci. USA 92: 1456±1460.
Dobrosotskaya I, Guy RK, James GL. 1997. MAGI-1, a membraneassociated guanylate kinase with a unique arrangement of
protein-protein interaction domains. J. Biol. Chem. 272:
31589±31597.
Dong LQ, Porter S, Hu DR, Liu F. 1998. Inhibition of hGrb10
binding to insulin receptor by functional domain-mediated
oligomerization. J. Biol. Chem. 273: 17720±17725.
Drozdov-Tikhomirov LN, Linde DM, Poroikov VV, Alexandrov AA,
Skurida GI. 2001. Molecular mechanisms of protein-protein
recognition: whether the surface placed charged residues
determine the recognition process? J. Biomol. Struct. Dyn.
19: 279±284.
Dunphy J, Linder ME. 1998. Signalling functions of protein
palmitoylation. Biochim. Biophys. Acta 1436: 245±261.
Eaton SR, Cody WL, Doherty AM, Holland DR, Panek RL, Lu GH,
Dahring TK, Rosa DR. 1998. Design of peptidomimetics that
inhibit the association of phosphatidylinisitol 3-kinase with
platelet-derived growth factor-beta receptor and possess
cellular activity. J. Med. Chem. 41: 4329±4342.
Ehring H. 1999. Hydrogen exchange/electrospray ionization mass
spectrometry studies of structural features of proteins and
protein/protein interactions. Anal. Biochem. 267: 252±259.
Eisenhaber F, Argos P. 1996. Hydrophobic regions on protein
surfaces: de®nition based on hydration shell structure and a
quick method for their computation. Protein Engng. 9: 1121±
1133.
Eisenmessers EZ, Post CB. 1998. Insights into phosphorilation
control of protein-protein association from the NMR structure
of a band 3 peptide inhibitor bound to glyceraldehyde-3phosphate dehydrogenase. Biochemistry 37: 867±877.
Eubanks S, Nguyen TL, Peyton D, Breslow E. 2000. Modulation of
J. Mol. Recognit. 2002; 15: 405–422
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
dimerization, binding, stability, and folding by mutation of
the neurophysin subunit interface. Biochemistry 39: 8085±
8094.
Eyster KM. 1998. Introduction to Signal Transduction. A primer
for untangling the web of intracellular messengers. Biochem.
Pharmac. 55: 1927±1938.
Fancy DA, Kodadek T. 1999. Chemistry for the analysis of protein±
protein interactions: rapid and ef®cient cross-linking triggered by long wavelength light. Proc. Natl Acad. Sci. USA 96:
6020±6024.
Findeis MA. 2000. Approaches to discovery and characterization
of inhibitors of amyloid beta-peptide polymerization. Biochim. Biophys. Acta 1502: 76±84.
Foti M, Cartier L, Piguet V, Lew DP, Carpentier JL, Trono D. Krause
KH. 1999. The HIV protein alters Ca2‡ signaling in myelomonocytic cells through SH3-mediated protein±protein interactions. J. Biol. Chem. 274: 24765±34772.
Freidinger RM. 1999. Nonpeptide ligands for peptide and protein
receptors. Curr. Opin. Chem. Biol. 3: 395±406.
Fretz H, Furet P, Garciaecheverria C, Rahuel J, Schoepfer J. 2000.
Structure-based design of compounds inhibiting Grb2-SH2
mediated protein±protein interactions in signal transduction
pathways. Curr. Pharm. Des. 6: 1777±1796.
Fuh G, Pisabarro MT, Li Y, Quan C, Lasky LA, Sidhu SS. 2000.
Analysis of PDZ domain-ligand interactions using carboxylterminal phage display. J. Biol. Chem. 275: 21486±21491.
Fujita T, Nakajima M, Inoue Y, Nose T, Shimohigashi Y. 1999. A
novel molecular design of thrombin receptor antagonist.
Bioorg. Med. Chem. Lett. 9: 1351±1356.
Furet P, Garcia-Echeverria C, Gay B, Schoepfer J, Zeller M, Rahuel
J. 1999. Structure-based design, synthesis, and X-ray crystallography of a high-af®nity antagonist of the -SH2 domain
containing an asparagine mimetic. J. Med. Chem. 42: 2358±
2363.
Furet P, Caravatti G, Denholm AA, Faessler A, Fretz H, GarciaEcheverria C, Gay B, Irving E, Press NJ, Rahuel J, Schoepfer
J, Walker CV. 2000. Structure-based design and synthesis of
phosphinate isosteres of phosphotyrosine for incorporation
in Grb2-SH2 domain inhibitors. Part 1. Bioorg. Med. Chem.
Lett. 10: 2337±2341.
Gabdoulline and Wade. 1999. J. Mol. Recognit. 12: 226±234.
Gao Y, Wu L, Luo JH, Guo R, Yang D, Zhang ZY, Burke TR Jr.
2000. Examination of novel non-phosphorus-containing
phosphotyrosyl mimetics against protein±tyrosine phosphatase-1B and demonstration of differential af®nities toward
Grb2 SH2 domains. Bioorg. Med. Chem. Lett. 10: 923±927.
Garciaecheverria C. 2001. Antagonists of the Src homology 2
(SH2) domains of Grb2, Src, Lck and ZAP-70. Curr. Med.
Chem. 8: 1589±1604.
Garnier C, Barbier P, Gilli R, Lopez C, Peyrot V, Briand C. 1998.
Head-shock protein 90 (hsp90) binds to tubulin dimer and
inhibits microtubule formation. Biochem. Biophys. Res.
Commun. 250: 414±419.
Gay B, Suarez S, Caravatti G, Furet P, Meyer T, Schoepfer J.
1999a. Selective GRB2 SH2 inhibitors as anti-Ras therapy. Int.
J. Cancer 83: 235±241.
Gay B, Suarez S, Weber C, Rahuel J, Fabbro D, Furet P, Schoepfer
J. 1999b. Effect of potent and selective inhibitors of Gbr2 SH2
domain on cell motility. J. Biol. Chem. 274: 23311±23315.
Ghosh I, Chmielewski J. 1998. A b-sheet peptide inhibitors of E47
dimerization and DNA binding. Chem. Biol. 5: 439±445.
Ghosh I, Issac R, Chmielewski J. 1999. Structure±function
relationship in a beta-sheet peptide inhibitor of E47 dimerization and DNA binding. Bioorg. Med. Chem. 7: 61±66.
Gibas CJ, Subramaniam S, McCammon JA, Braden BC, Poljak
RJ. 1997. pH dependence of antibody/lysozyme complexation. Biochemistry 36: 15599±15614.
Glaser RW, Hausdorf G. 1996. Binding kinetics of an antibody
against HIV p24 core protein measured with real-time
biomolecular interaction analysis suggest a slow conformation change in antigen p24. J. Immunol. Meth. 189: 1±14.
Gloss LM, Matthews CR. 1998. The barriers in the bimolecular
and unimolecular folding reactions of the dimeric core
domain of Escherichia coli Trp repressor are dominated by
enthalpic contributions. Biochemistry 37: 16000±16010.
Copyright # 2002 John Wiley & Sons, Ltd.
417
Gomez J, Freire E. 1995. Thermodynamic mapping of the
inhibitor site of the aspartic protease endothiapepsin. J.
Mol. Biol. 252: 337±350.
Gomez-Moreno C, Martinez-Julvez M, Medina M, Hurley JK,
Tollin G. 1998. Protein±protein interaction in electron transfer
reactions: the ferredoxin/¯avodoxin/ferredoxin: NADP ‡
reductase system from Anabaena. Biochimie 80: 837±846.
Gong XS, Wen JQ, Fisher NE, Young S, Howe CJ, Bendall DS,
Gray JC. 2000. The role of individual lysine residues in the
basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro. Eur. J. Biochem. 267: 3461±
3468.
Gotz C, Scholtes P, Prowald A, Schuster N, Nastainczyk W,
Montenarh M. 1999. Protein kinase CK2 interacts with a multiprotein binding domain of p53. Mol. Cell Biochem. 191: 111±
120.
Grant GA, Xu XL, Hu ZQ. 2000. Removal of the tryptophan 139
side chain in Escherichia coli D-3-phosphoglycerate dehydrogenase produces a dimeric enzyme without cooperative
effects. Arch. Biochem. Biophys. 375: 171±174.
Grucza RA, Bradshaw JM, Mitaxov V, Waksman G. 2000. Role of
electrostatic interactions in SH2 domain recognition: saltdependence of tyrosyl-phosphorylated peptide binding to
the tandem SH2 domain of the Syk kinase and the single SH2
domain of the Src kinase. Biochemistry 39: 10072±10081.
Hart CP, Martin JE, Reed MA, Keval AA, Pustelnik MJ, Northrop
JP, Grove JR. 1999. Potent inhibitory ligands of the GRB2 SH2
domain from recombinant peptide libraries. Cell Signal. 11:
453±464.
Herrmann H, Aebi U. 1998. Intermediate ®lament assembly:
®brillogenesis is driven by decisive dimer±dimer interactions. Curr. Opin. Struct. Biol. 8: 177±185.
Hilpert K, Behlke J, Scholz C, Misselwitz R, Schneider-Mergener
J, Hohne W. 1999. Interaction of the caspid protein p24 (HIV1) with sequence-derived peptides. In¯uence on p24 dimerization. Virology 254: 6±10.
Ho SN, Biggar SR, Spencer DM, Schreiber SL, Crabtree GR. 1996.
Dimeric ligands de®ne a role for transcriptional activation
domains in reinitiation. Nature 382: 822±826.
Hoekstra WJ, Hulshizer BL, McComsey DF, Andrade-Gordon P,
Kauffman JA, Addo MF, Oksenberg D, Scarborough RM,
Maryanoff BE. 1998. Thrombin receptor (PAR-1) antagonists.
Heterocycle-based peptidomimetics of the SFLLR agonist
motif. Bioorg. Med. Chem. Lett. 8: 1649±1654.
Holwerda B. 1999. Activity in monomers of human cytomegalovirus protease. Biochem. Biophys. Res. Commun. 259: 370±
373.
Hu JC, Kornacker MG, Hochschild A. 2000a. Escherichia coli oneand two-hybrid systems for the analysis and identi®cation of
protein±protein interactions. Methods 20: 80±94.
Hu ZJ, Ma BY, Wolfson H, Nussinov R. 2000b. Conservation of
polar residues as hot spots at protein interfaces. Proteins 39:
331±342.
Huang HW, Chen WC, Wu CY, Yu HC, Lin WY, Chen ST, Wang KT.
1997. Kinetic studies of the inhibitory effects of propeptides
subtilisin BPN' and Carlsberg to bacterial serine proteases.
Protein Engng 10: 1227±1233.
Hubbard SJ, Argos P. 1994. Cavities and packing at protein
interfaces. Protein Sci. 3: 2194±2206.
Imai Y, Moralez A, Andag U, Clarke JB, Busby WH, Clemmons
DR. 2000. Substitutions for hydrophobic amino acids in the
N-terminal domains of IGFBP-3 and-5 markedly reduce IGF-I
binding and alter their biologic actions. J. Biol. Chem. 275:
18188±18194.
Ivanov YD, Kanaeva IP, Kuznetsov VY, Lehnerer M, Schulze J,
Hlavica P, Archakov AI. 1999a. The optical biosensor studies
on the role of hydrophobic tails of NADPH-cytochrome P450
reductase and cytochromes P450 2B4 and b5 upon productive complex formation within a monomeric reconstituted
system. Arch. Biochem. Biophys. 362: 87±93.
Ivanov YD, Usanov SA, Archakov AI. 1999b. The optical
biosensor studies on the productive complex formation
between the components of cytochrome P450scc dependent
monooxygenase system. Biochem. Mol. Biol. Int. 47: 327±
336.
J. Mol. Recognit. 2002; 15: 405–422
418
A. V. VESELOVSKY ET AL.
Ivanov Yu, Kanaeva I, Archakov A. 2000. Optical biosensor studys
of ternary complex formation in a cytochrome P450 2B4
system. Biochem. Biophys. Res. Commun. 273: 750±752.
Ivanov YD, Kanaeva IP, Karuzina IP, Archakov AI, Hui Bon Hoa G,
Sligar SG. 2001. Molecular recognition in the P450cam
monooxygenase system: direct monitoring of protein±protein interaction by using optical biosensor. Arch. Biochem.
Biophys. 391: 255±264.
Jackson RM. 1999. Comparison of protein±protein interactions in
serine protease±inhibitor and antibody±antigen complexes:
Implications for the protein docking problem. Protein Sci. 8:
603±613.
Janin J. 1995. Principles of protein±protein recognition from
structure to thermodynamics. Biochimie 77: 497±505.
Janin J. 1999. Wet and dry interfaces: the role of solvent in
protein±protein and protein±DNA recognition. Struct. Fold.
Des. 7: 277±279.
Janin J. 2000. Kinetics and thermodynamics of protein±protein
interactions. In Protein±Protein Recognition. Oxford University Press: New York; 1±32.
Jones S, Thornton JM. 1996. Principles of protein±protein
interactions. Proc. Natl Acad. Sci. USA 93: 13±20.
Jones S, Thornton JM. 2000. Analysis and classi®cation of
protein±protein interactions from a structural perspective. In
Protein±Protein Recognition. Oxford University Press: New
York; 33±59.
Ju H, Venema VJ, Marrero MB, Venema RC. 1998. Inhibitory
interactions of the brakinin B2 receptor with endothelial
nitric-oxide synthase. J. Biol. Chem. 273: 24025±24029.
Kameshita I, Ishida A, Fujisawa H. 1998. Analysis of protein±
protein interaction by two-dimensional af®nity electrophoresis. Anal. Biochem. 262: 90±92.
Kato M, Iwashita T, Takeda K, Akhand AA, Liu W, Yoshihara M,
Hossain K, Wu J, Du J, Oh C, Kawamoto Y, Suzuki H,
Takahashi M, Nakashima I. 2000. Ultraviolet light induces
redox reaction-mediated dimerization and superactivation of
oncogenic Ret tyrosine kinases. Mol. Biol. Cell 11: 93±101.
Keenan T, Yaeger DR, Courage NL, Rollins CT, Pavone ME, Rivera
VM, Yang W, Guo T, Amara JF, Clackson T, Gilman M, Holt
DA. 1998. Synthesis and activity of bivalent FKBP12 ligands
for the regulated dimerization of proteins. Bioorg. Med.
Chem. 6: 1309±1335.
Kehoe JW, Bertozzi CR. 2000. Tyrosine sulfation: a modulator of
extracellular protein±protein interactions. Chem. Biol. 7: R57±
R61.
Kim HK, Nam JY, Han MY, Lee EK, Choi JD, Bok SH, Kwon BM.
1999. Actinomycin D as a novel SH2 domain ligand inhibits
Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3)
and SAA (hEGFR-overexpressed NIH3T3) cells. FEBS Lett.
453: 174±178.
Kimura SR, Brower RC, Vajda S, Camacho CJ. 2001. Dynamical
view of the positions of key side chains in protein±protein
recognition. Biophys. J. 80: 635±642.
Kiselyova OI, Yaminsky IV, Ivanov YuD, Kanaeva IP, Kuznetsov
VYu, Archakov AI. 1999. AFM study of membrane proteins,
cytochrome P450 2B4 and NADPH-cytochrome P450 reductase, and of their complex formation. Arch. Biochem.
Biophys. 371: 1±7.
Klemm JD, Schreiber SL, Crabtree GR. 1998. Dimerization as a
regulatory mechanism in signal transduction. A. Rev.
Immunol. 16: 569±592.
Kojima S, Minagawa T, Miura K. 1998. Tetriary structure
formation in the propeptide of subtilisin BPN' by successive
amino acid replacements. J. Mol. Biol. 277: 1007±1013.
Koltzscher M, Gerke V. 2000. Identi®cation of hydrophobic amino
acid residues involved in the formation of S100P homodimers in vivo. Biochemistry 39: 9533±9539.
Kopytek SJ, Standaert RF, Dyer JCD, Hu JC. 2000. Chemically
induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate. Chem. Biol. 7: 313±321.
Kozlov G, Gehring K, Ekiel I. 2000. Solution structure of the PDZ2
domain from human phosphatase hPTP1E and its interaction
with C-terminal peptides from the Fas receptors. Biochemistry 39: 2572±2580.
Kuhlmann UC, Pommer AJ, Moore GR, James R, Kleanthous C.
Copyright # 2002 John Wiley & Sons, Ltd.
2000. Speci®city in protein±protein interactions: The structural basis for dual recognition in endonuclease colicinimmunity protein complexes. J. Mol. Biol. 301: 1163±1178.
Laitinen OH, Marttila AT, Airenne KJ, Kulik T, Livnah O, Bayer EA,
Wilchek M, Kulomaa MS. 2001. Biotin induces tetramerization of a recombinant monomeric avidin Ð A model for
protein±protein interactions. J. Biol. Chem. 276: 8219±8224.
Larsen TA, Olson AJ, Goodsell DS. 1998. Morphology of protein±
protein interfaces. Structure 6: 421±427.
Le F, Stomski F, Woodcock JM, Lopez AF, Gonda TJ. 2000. The
role of disul®de-linked dimerization in interleukin-3 receptor
signaling and biological activity. J. Biol. Chem. 275: 5124±
5130.
Leavitt S, Freire E. 2001. Direct measurement of protein binding
energetics by isothermal titration calorimetry. Curr. Opin.
Struct. Biol. 11: 560±566.
Lebl M. 1999. New technique for high-throughput synthesis.
Bioorg. Med. Chem. Lett. 9: 1305±1310.
Lehnerer M, Schulze J, Pernecky SJ, Lewis DFV, Eulitz M, Hlavica
P. 1998. In¯uence of mutation of the amino-terminal signal
anchor sequence of cytochrome P450 2B4 on the enzyme
structure and electron transfer processes. J. Biochem. 124:
396±403.
Li S, Satoh T, Korngold R, Huang Z. 1998. CD4 dimerization and
oligomerization: implications for T-cell function and structure-based drug design. Immunol. Today 19: 455±62.
Li YL, Li HM, Smithgill SJ, Mariuzza RA. 2000. Three-dimensional
structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63. Biochemistry 39:
6296±6309.
Lijnzaad P, Argos P. 1997. Hydrophobic patches on protein
subunit interfaces: Characteristics and prediction. Proteins
28: 333±343.
Lin CT, Kuo TJ, Shaw JF, Kao MC. 1999. Characterization of the
dimer±monomer equilibrium of the papaya copper/zinc
superoxide dismutase and its equilibrium shift by a single
amino acid mutation. J. Agric. Food Chem. 47: 2944±2949.
Liu JO. 1999. Recruitment of proteins to modulate protein±
protein interactions. Chem. Biol. 6: R213±R216.
Liuzzi M, Deziel R, Moss N, Beaulieu P, Bonneau AM, Bousquet C,
Chafouleas JG, Garneau M, Jaramillo J, Krogsrud RL et al.
1994. A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo. Nature 372: 695±
698.
Lo Conte L, Chothia C, Janin J. 1999. The atomic structure of
protein±protein recognition sites. J. Mol. Biol. 285: 2177±
2198.
Lomax ME, Barnes DM, Hupp TR, Picksley SM, Camplejohn RS.
1998. Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family
members. Oncogene 17: 643±649.
Lou YC, Lung FD, Pai MT, Tzeng SR, Wei SY, Roller PP, Cheng JW.
1999. Solution structure and dynamics of G1TE, a nonphosphorylated cyclic peptide inhibitor for the Grb2 SH2 domain.
Arch. Biochem. Biophys. 372: 309±314.
Loughney DA, Murray WV, Jolliffe LK. 1999. Application of virtual
screening tools to a protein±protein interaction: Database
mining studies on the growth hormone receptor. Med. Chem.
Res. 9: 579±591.
MacCorkle RA, Freeman KW, Spencer DM. 1998. Synthetic
activation of caspases: arti®cial death switches. Proc. Natl
Acad. Sci. USA 95: 3655±3660.
Markus M, Benezra R. 1999. Two isoforms of protein disul®de
isomerase alter the dimerization status of E2A proteins by a
redox mechanism. J. Biol. Chem. 274: 1040±1049.
Maroun RG, Gayet S, Benleulmi MS, Porumb H, Zargarian L,
Merad H, Leh H, Mouscadet JF, Troalen FR, Fermandjian S.
2001. Peptide inhibitors of HIV-1 integrase dissociate the
enzyme oligomers. Biochemistry 40: 13840±13848.
Marrone TJ, Briggs JM, McCammon JA. 1997. Structure-based
drug design: computational advances. A. Rev. Pharmac.
Toxicol. 37: 71±90.
Martin C, Hartley R, Mauguen Y. 1999a. X-ray structural analysis
of compensating mutations at the barnase-barstar interface.
FEBS Lett. 452: 128±132.
J. Mol. Recognit. 2002; 15: 405–422
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
Martin DC, Pastralandis SC, Kantrowitz ER. 1999b. Amino acid
substitutions at the subunit interface of dimeric Escherichia
coli alkaline phosphatase cause reduced structural stability.
Protein Sci. 8: 1152±1159.
Marusawa H, Setoi H, Kuroda A, Sawada A, Seki J, Motoyama Y,
Tanaka H. 1999. Synthesis and biological activity of 4-methyl3,5-dioxane derivatives as thromboxane A2 receptor antagonists. Bioorg. Med. Chem. 7: 2635±2645.
Massova I, Kollman PA. 1999. Computational alanine scanning to
probe protein±protein interactions: A novel approach to
evaluate binding free energies. J. Am. Chem. Soc. 121:
8133±8143.
Mateu MG, Fersht AR. 1998. Nine hydrophobic side chains are
key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization
domain. EMBO J. 17: 2748±2758.
Mathews FS, Mauk AG, Moore GR. 2000. Protein±protein
complexes formed by electron transfer proteins. In Protein±
Protein Recognition. Oxford University Press: New York; 60±
101.
McCammon JA. 1998. Theory of biomolecular recognition. Curr.
Opin. Struct. Biol. 8: 245±249.
McCoy AJ, Chandana EV, Colman PM. 1997. Electrostatic
complementarity at protein/protein interfaces. J. Mol. Biol.
268: 570±584.
McDonnell JM. 2001. Surface plasmon resonance: towards an
understanding of the mechanisms of biological molecular
recognition. Curr. Opin. Chem. Biol. 5: 572±577.
McInnes C, Grothe S, O'Connor-McCourt M, Sykes BD. 2000.
NMR study of the differential contributions of residues of
transforming growth factor alpha to association with its
receptor. Protein Engng 13: 143±147.
Metcalf CA, Eyermann CJ, Bohacek RS, Haraldson CA, Varkhedkar VM, Lynch BA, Bartlett C, Violette SM, Sawyer TK. 2000.
Structure-based design and solid-phase parallel synthesis of
phosphorylated nonpeptides to explore hydrophobic binding
at the Src SH2 domain. J. Comb. Chem. 2: 305±313.
Michnick SW. 2000. Chemical biology beyond binary codes.
Chem. Biol. 7: R217±R221.
Morris MC, Robert-Hebmann V, Chaloin L, Mery J, Heitz F,
Devaux C, Goody RS, Divita G. 1999. A new potent HIV-1
reverse transcriptase inhibitor. A synthetic peptide derived
from the interface subunit domains. J. Biol. Chem. 274:
24941±24946.
Muegge I, Schweins T, Warshel A. 1998. Electrostatic contributions to protein±protein binding af®nities: application to Rap/
Raf interaction. Proteins 30: 407±423.
Murray-Rust J, McLeod AN, Blundell TL, Wood SP. 1992.
Structure and evolution of insulins: implications for receptor
binding. Bioessays 14: 325±331.
Nefzy A, Dooley C, Ostresh JM, Houghten RA. 1998. Combinatorial chemistry: from peptides and peptidomimetics to small
organic and heterocyclidc compounds. Bioorg. Med. Chem.
Lett. 8: 2273±2278.
Nguyen JT, Porter M, Amoui M, Miller WT, Zuckermann RN, Lim
WA. 2000. Improving SH3 domain ligand selectivity using a
non-natural scaffold. Chem. Biol. 7: 463±473.
Nicole P, Lins L, Rouyer-Fessard C, Drouot C, Fulcrand P, Thomas
A, Couvineau A, Martinez J, Brasseur R, Laburthe M. 2000.
Identi®cation of key residues for interaction of vasoactive
intestinal peptide with human VPAC(1) and VPAC(2) receptors and development of a highly selective VPAC(1) receptor
agonist. Alanine scanning and molecular modeling of the
peptide. J. Biol. Chem. 275: 24003±24012.
Niimi T, Orita M, Okazawaigarashi M, Sakashita H, Kikuchi K, Ball
E, Ichikawa A, Yamagiwa Y, Sakamoto S, Tanaka A,
Tsukamoto S, Fujita S, Tatsuta K, Maeda Y, Chikauchi K.
2001. Design and synthesis of non-peptidic inhibitors for the
Syk C-terminal SH2 domain based on structure-based insilico screening. J. Med. Chem. 44: 4737±4740.
Nordhoff A, Tziatzios C, van den Broek JA, Schott MK, Kalbitzer
HR, Becker K, Schubert D, Schirmer RH. 1997. Denaturation
and reactivation of dimeric human glutation reductase Ð an
assay for folding inhibitors. Eur. J. Biochem. 245: 273±282.
Norton JD, Deed RW, Craggs G, Sablitzky F. 1998. Id helix±loop±
Copyright # 2002 John Wiley & Sons, Ltd.
419
helix proteins in cell growth and differentiation. Trends Cell
Biol. 8: 58±65.
Okada D. 1998. Tetrahydrobiopterin-dependent stabilization of
neuronal nitric oxide synthase dimer reduces susceptibility
to phosphorylation by protein kinase C in vitro. FEBS Lett.
434: 261±264.
Omata Y, Dai RK, Smith SV, Robinson RC, Friedman FK. 2000.
Synthetic peptide mimics of a predicted topographical
interaction surface: the cytochrome P4502B1 recognition
domain for NADPH-cytochrome P450 reductase. J. Protein
Chem. 19: 23±32.
Onofri F, Giovedi S, Kao HT, Valtorta F, Borbone LB, De Camilli P,
Greengard P, Benfenati F. 2000. Speci®city of the binding of
synapsin I to Src homology 3 domains. J. Biol. Chem. 275:
29857±29867.
Ortiz MA, Light J, Maki RA, Assa-Munt N. 1999. Mutation analysis
of the Pip interaction domain reveals critical residues for
protein±protein interactions. Proc. Natl Acad. Sci. USA 96:
2740±2745.
Otzen DE, Fersht AR. 1999. Analysis of protein±protein interactions by mutagenesis: direct versus indirect effects. Protein
Engng 12: 41±45.
Overton MC, Blumer KJ. 2000. G-protein-coupled receptors
function as oligomers in vivo. Curr. Biol. 10: 341±344.
Pace AJ, Gama L, Breitwieser GE. 1999. Dimerization of the
calcium-sensing receptor occurs within the extracellular
domain and is eliminated by Cys → Ser mutations at
Cys(101) and Cys(236). J. Biol. Chem. 274: 11629±11634.
Pacofsky GJ, Lackey K, Alligood KJ, Berman J, Charifson PS,
Crosby RM, Dorsey GF Jr, Feldman PL, Gilmer TM, Hummel
CW, Jordan SR, Mohr C, Shewchuk LM, Sternbach DD,
Rodriguez M. 1998. Potent dipeptide inhibitors of the pp60csrc SH2 domain. J. Med. Chem. 41: 1894±1908.
Pardanani A, Gambacurta A, Ascoli F, Royer WE. 1998. Mutational destabilization of the critical interface water cluster in
Scapharca dimeric hemoglobin. Structural basis for altered
allosteric activity. J. Mol. Biol. 284: 729±739.
Park SH, Raines RT. 1997. Green ¯uorescent protein as a signal
for protein±protein interactions. Protein Sci. 6: 2344±2349.
Park SH, Raines RT. 2000. Genetic selection for dissociative
inhibitors of designated protein±protein interactions. Nat.
Biotechnol. 18: 847±851.
Parker SL, Parker MS. 2000. FMRF amides exert a unique
modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors. Can. J. Physiol. Pharmac. 78:
150±161.
Pawson T. 1995. Protein modules and signalling networks.
Nature 373: 573±580.
Pawson T, Gish GD, Nah P. 2001. SH2 domains, interaction
modules and cellular wiring. Trends Cell Biol. 11: 504±511.
Pepys MB, Herbert J, Hutchinson WL, Tennent GA, Lachmann HJ,
Gallimore JR, Lovat LB, Bartfai T, Alanine A, Hertel C,
Hoffmann T, Jakob-Roetne R, Norcross RD, Kemp JA,
Yamamura K, Suzuki M, Taylor GW, Murray S, Thompson
D, Purvis A, Kolstoe S, Wood SP, Hawkins PN. 2002. Targeted
pharmacological depletion of serum amyloid P component
for treatment of human amyloidosis. Nature 417: 254±259.
Pons J, Rajpal A, Kirsch JF. 1999. Energetic analysis of an
antigen/antibody interface: Alanine scanning mutagenesis
and double mutant cycles on the HyHEL-10/lysozyme interaction. Protein Sci. 8: 958±968.
Prasanna V, Bhattacharjya S, Balaram P. 1998. Synthetic interface
peptides as inactivators of multimeric enzymes: inhibitory
and conformational properties of three fragments from
Lactobacillus casei thymidylate synthase. Biochemistry 37:
6883±6893.
Prevelige PE Jr. 1998. Inhibiting virus-capsid assembly by
altering the polymerisation pathway. Trends Biotechnol. 16:
61±65.
Rafferty SP, Boyington JC, Kulansky R, Sun PD, Malech HL. 1999.
Stoichiometric arginine binding in the oxygenase domain of
inducible nitric oxide synthase requires a single molecule of
tetrahydrobiopterin per dimer. Biochem. Biophys. Res.
Commun. 257: 344±347.
Ramadevi N, Rodrigues J, Roy P. 1998. A leucine zipper-like
J. Mol. Recognit. 2002; 15: 405–422
420
A. V. VESELOVSKY ET AL.
domain is essential for dimerization and encapsidation of
bluetongue virus nucleocapsid protein VP4. J. Virol. 72:
2983±2990.
Ratovitski EA, Alam MR, Quick RK, McMillan A, Bao C, Kozlovsky
C, Hand TA, Johnson RC, Mains RE, Eipper BA, Lowenstein
CJ. 1999. Kalirin inhibition of inducible nitric-oxide synthase.
J. Biol. Chem. 274: 993±999.
Rich RL, Myszka DG. 2001. Survey of the year 2000 commercial
optical biosensor literature. J. Mol. Recognit. 14: 273±294.
Rickles RJ, Henry PA, Guan W, Azimioara M, Shakespeare WC,
Violette S, Zoller MJ. 1998. A novel mechanism-based
mammalian cell assay for the identi®cation of SH2-domainspeci®c protein±protein inhibitors. Chem. Biol. 5: 529±538.
Ripka AS, Rich DH. 1998. Peptidomimetic design. Curr. Opin.
Chem. Biol. 2: 441±452.
Rodriguezcrespo I, Straub W, Gavilanes F, Demontellano PRO.
1998. Binding of dynein light chain (PIN) to neuronal nitric
oxide synthase in the absence of inhibition. Arch. Biochem.
Biophys. 359: 297±304.
Rohrer SP, Birzin ET, Mosley RT, Berk SC, Hutchins SM, Shen DM, Xiong Y, Hayes EC, Parmar RM, Foor F, Mitra SW,
Degrado SJ, Shu M, Klopp JM, Cai SJ, Blake A, Chan WW,
Pasternak A, Yang L, Patchett AA, Smith RG, Chapman KT,
Schaeffer JM. 1998. Rapid identi®cation of subtype-selective
agonists of the somastatin receptor through combinatorial
chemistry. Science 282: 737±740.
Rudert F, Woltering C, Frisch C, Rottenberger C, Ilag LL. 1998. A
phage-based system to select multiple protein±protein
interactions simultaneously from combinatorial libraries.
FEBS Lett. 440: 135±140.
Rudiger AH, Rudiger M, Carl UD, Chakraborty T, Roepstorff P,
Wehland J. 1999. Af®nity mass spectrometry-based approaches for the analysis of protein±protein interaction and
complex mixtures of peptide±ligands. Anal. Biochem. 275:
162±170.
Saarela J, Laine M, Tikkanen R, Oinonen C, Jalanko A, Rouvinen
J, Peltonen L. 1998. Activation and oligomerization of
aspartylglucosaminidase. J. Biol. Chem. 273: 25320±25328.
Saito S, Watabe S, Ozaki H, Kobayshi M, Suzuki T, Kobayshi H,
Fusetani N, Karaki H. 1998. Actin-depolymerizing effect of
dimeric macrolides, bistheonellide A and swinholide A. J.
Biochem. 123: 571±578.
Salahpour A, Angers S, Bouvier M. 2000. Functional signi®cance
of oligomerization of G-protein-coupled receptors. Trends
Endocrinol. Metab. 11: 163±168.
Salzmann M, Bachmann MF. 1998. The role of T cell receptor
dimerization for T cell antagonism and T cell speci®city. Mol.
Immunol. 35: 271±277.
Sasvari Z, Asboth B. 1998. Formation of disul®de-bridges dimers
during theroinactivation of glucoamylase from Aspergillus
niger. Enzyme Microb. Technol. 22: 466±470.
Schenkman JB, Jansson I. 1999. Interactions between cytochrome P450 and cytochrome b5. Drug Metab. Rev. 31: 351±
364.
Schoepfer J, Gay B, Caravatti G, Garcia-Echeverria C, Fretz H,
Rahuel J, Furet P. 1998. Structure-based design of peptidomimetic ligands of the Grb2-SH2 domain. Bioorg. Med.
Chem. Lett. 8: 2865±2870.
Schoepfer J, Fretz H, Gay B, Furet P, Garcia-Echeverria C, End N,
Caravatti G. 1999. Highly potent inhibitors of the Grb2-SH2
domain. Bioorg. Med. Chem. Lett. 9: 221±226.
Schramm HJ, Derosny E, Reboudravaux M, Buttner J, Dick A,
Schramm W. 1999. Lipopeptides as dimerization inhibitors of
HIV-1 protease. Biol. Chem. 380: 593±596.
Schreiber G, Frisch C, Fersht AR. 1997. The role of Glu73 of
barnase in catalysis and the binding of barstar. J. Mol. Biol.
270: 111±122.
Schuck P, Millar DB, Kortt AA. 1998. Determination of binding
constants by equilibrium titration with circulating sample in a
surface plasmon resonance biosensor. Anal. Biochem. 265:
79±91.
Schumacher C, Wang H, Honer C, Ding W, Koehn J, Lawrence Q,
Coulis CM, Wang LL, Ballinger D, Bowen BR, Wagner S. 2000.
The SCAN domain mediates selective oligomerization. J.
Biol. Chem. 275: 17173±17179.
Copyright # 2002 John Wiley & Sons, Ltd.
Scott S, Dong FM, Kisterswoike B, Mullerhill B. 2000. Dimerisation mutants of Lac repressor. II. A singe amino acid
substitution, D278L, changes the speci®city of dimerisation.
J. Mol. Biol. 296: 673±684.
Selkoe DJ. 1998. The cell biology of b-amyloid precursor protein
and presenilin in Alzheimer's disease. Trends Cell Biol. 8:
443±457.
Sengchanthalangsy LL, Datta S, Huang DB, Anderson E, Braswell
EH, Ghosh G. 1999. Characterization of the dimer interface of
transcription factor NF kappa B p50 homodimer. J. Mol. Biol.
289: 1029±1040.
Sennequier N, Wolan D, Stuehr DJ. 1999. Antifungal imidazoles
block assembly of inducible NO synthase into an active
dimer. J. Biol. Chem. 274: 930±938.
Shakespeare WC. 2001. SH2 domain inhibition: a problem
solved? Curr. Opin. Chem. Biol. 5: 409±415.
Shakespeare W, Yang M, Bohacek R, Cerasoli F, Stebbins K,
Sundaramoorthi R, Azimioara M, Vu C, Pradeepan S, Metcalf
C III, Haraldson C, Merry T, Dalgarno D, Narula S, Hatada M,
Lu X, van Schravendijk MR, Adams S, Violette S, Smith J,
Guan W, Bartlett C, Herson J, Iuliucci J, Weigele M, Sawyer T.
2000. Structure-based design of an osteoclast-selective,
nonpeptide Src homology 2 inhibitor with in vivo antiresorptive activity. Proc. Natl Acad. Sci. USA 97: 9373±9378.
Sheinerman FB, Norel R, Honig B. 2000. Electrostatic aspects of
protein±protein interactions. Curr. Opin. Struct. Biol. 10: 153±
159.
Shima S, Tziatzios C, Schubert D, Fukada H, Takahashi K, Ermler
U, Thauer RK. 1998. Lyotropic-salt induced changes in
monomer/dimer/tetramer association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri in relation to the activity and thermostability of the
enzyme. Eur. J. Biochem. 258: 85±92.
Shultz MD, Chmielewski J. 1997. Hydrophobicity versus activity
in crosslinked interfacial peptide inhibitor of HIV-1 protease.
Tetrahedron 8: 3881±3886.
Shultz MD, Chmielewski J. 1999. Probing the role of interfacial
residues in a dimerization inhibitor of HIV-1 protease. Bioorg.
Med. Chem. Lett. 9: 2431±2436.
Shultz MD, Bowman MJ, Ham YW, Zhao XM, Tora G, Chmielewski J. 2000. Small-molecule inhibitors of HIV-1 protease
dimerization derived from cross-linked interfacial peptides.
Angew. Chem. 39: 2710±2713.
Singh SK, Maithal K, Balaram H, Balaram P. 2001. Synthetic
peptides as inactivators of multimeric enzymes: inhibition of
Plasmodium falciparum triosephosphate isomerase by interface peptides. FEBS Lett. 501: 19±23.
Sloan DJ, Hellinga HW. 1998. Structure-based engineering of
environmentally sensitive ¯uorophores for monitoring protein±protein interactions. Protein Engng 11: 819±823.
Smith KM, Vanetten RA. 2001. Activation of c-Abl kinase activity
and transformation by a chemical inducer of dimerization. J.
Biol. Chem. 276: 24372±24379.
Smyczynski C, Derancourt J, Chaussepied P. 2000. Regulation of
ncd by oligomeric state of tubulin. J. Mol. Biol. 295: 325±336.
Song MC, Rajesh S, Hayashi Y, Kiso Y. 2001. Design and
synthesis of new inhibitors of HIV-1 protease dimerization
with conformationally constrained templates. Bioorg. Med.
Chem. Lett. 11: 2465±2468.
Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH,
Crompton A, Chan AC, Anderson JM, Cantley LC. 1997.
Recognition of unique carboxyl-terminal motifs by distinct
PDZ domains. Science 275: 73±77.
Sourgen F, Maroun RG, Frere V, Bouziane M, Auclair C, Troalen F,
Fermandjian S. 1996. A synthetic peptide from the human
immunode®ciency virus type-1 integrase exibits coiled-ciol
properties and interfaces with the in vitro integration activity
of the enzyme. Eur. J. Biochem. 240: 765±773.
Souroujon MC, Mochly-Rosen D. 1998. Peptide modulators of
protein±protein interactions in intracellular signaling. Nat.
Biotechnol. 16: 919±924.
Srivastava AK, Sauer RT. 2000. Evidence for partial secondary
structure formation in the transition state for Arc repressor
refolding and dimerization. Biochemistry 39: 8308±8314.
Stenberg G, Abdalla AM, Mannervik B. 2000. Tyrosine 50 at the
J. Mol. Recognit. 2002; 15: 405–422
PROTEIN–PROTEIN INTERACTIONS AND DRUGS
subunit interface of dimeric human glutathione transferase
P1-1 is a structural key residue for modulating protein
stability and catalytic function. Biochem. Biophys. Res.
Commun. 271: 59±63.
Stevens JM, Armstrong RN, Dirr HW. 2000. Electrostatic interactions affecting the active site of class Sigma glutathione Stransferase. Biochem. J. 347: 193±197.
Stigers KD, Soth MJ, Nowick JS. 1999. Designed molecules that
fold to mimic protein secondary structures. Curr. Opin.
Chem. Biol. 3: 714±723.
Stites WE. 1997. Protein±protein interactions: interface structure,
binding thermodynamics, and mutational analysis. Chem.
Rev. 97: 1233±1250.
Sundberg EJ, Mariuzza RA. 2000. Luxury accommodations: the
expanding role of structural plasticity in protein±protein
interactions. Struct. Fold. Des. 8: R137±R142.
Sundberg EJ, Urrutia M, Braden BC, Isern J, Tsuchiya D, Fields
BA, Malchiodi EL, Tormo J, Schwarz FP, Mariuzza RA. 2000.
Estimation of the hydrophobic effect in an antigen-antibody
protein±protein interface. Biochemistry 39: 15375±15387.
Tamura A, Privalov PL. 1997. The entropy cost of protein
association. J. Mol. Biol. 273: 1048±1060.
Taniguchi H. 1999. Protein myristoylation in protein-lipid and
protein±protein interactions. Biophys. Chem. 82: 129±137.
Tarasova NI, Rice WG, Michejda CJ. 1999. Inhibition of G-proteincoupled receptor function by disruption of transmembrane
domain interactions. J. Biol. Chem. 274: 34911±34915.
Thomas MC, Ballantine SP, Bethell SS, Bains S, Kellam P, Delves
CJ. 1998. Single amino acid substitutions disrupt tetramer
formation in the dihydroneopterin aldolase enzyme of
Pneumocystis carinii. Biochemistry 37: 11629±11636.
Tochio H, Zhang Q, Mandal P, Li M, Zhang M. 1999. Solution
structure of neuronal nitric oxide synthase PDZ domain
complexed with an associated peptide. Nat. Struct. Biol. 6:
417±421.
Tsai C-J, Lin SL, Wolfson HJ, Nissinov R. 1996. Protein±protein
interfaces: Architectures and interactions in protein±protein
interfaces and in protein cores. Their similarities and
differences. Crit. Rev. Biochem. Mol. Biol. 31: 127±152.
Tsai C-J, Lin SL, Wolfson HJ, Nissinov R. 1997a. Studies of
protein±protein interfaces: a statistical analysis of hydrophobic effect. Protein Sci. 6: 53±64.
Tsai C-J, Xu D, Nissinov R. 1997b. Structural motifs at protein±
protein interfaces: protein cores versus two-state and threestate model complexes. Protein Sci. 6: 1797±1809.
Tsuda T, Ikeda Y, Taniguchi N. 2000. The Asn-420-linked sugar
chain in human epidermal growth factor receptor suppresses
ligand-independent spontaneous oligomerizationÐpossible
role of a speci®c chain in controllable receptor activation. J.
Biol. Chem. 275: 21988±21994.
Ubarretxena-Belandia I, Hozeman L, Vanderbrinkvanderlaan E,
Pap EHM, Egmond MR, Verheij HM, Dekker N. 1999. Outer
membrane phospholipase A is dimeric in phospholipid
bilayers: a cross-linking and ¯uorescence resonance energy
transfer study. Biochemistry 38: 7398±7405.
Ulysse LG, Chmielewski J. 1998. Restricting the ¯exibility of
crosslinked, interfacial peptide inhibitors of HIV-1 protease.
Bioorg. Med. Chem. Lett. 8: 3281±3286.
Usui T, Kondoh M, Mayumi T, Osada H. 1998. Tryptostatin A, a
speci®c and novel inhibitor of microtubule assembly.
Biochem. J. 333: 543±548.
Valentemesquita VL, Botelho MM, Ferreira ST. 1998. Pressureinduced subunit dissociation and unfolding of dimeric betalactoglobulin. Biophys. J. 75: 471±476.
Vaughan CK, Buckle AM, Fersht AR. 1999. Structural response to
mutation at a protein±protein interface. J. Mol. Biol. 286:
1487±1506.
Velev OD, Kaler EW, Lenhoff AM. 1998. Protein interactions in
solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen. Biophys. J. 75:
2682±2697.
Vergnon AL, Chu YH. 1999. Electrophoretic methods for studying
protein±protein interactions. Methods 19: 270±277.
Viani MB, Pietrasanta LI, Thompson JB, Chand A, Gebeshuber IC,
Kindt JH, Richter M, Hansma HG, Hansma PK. 2000. Probing
Copyright # 2002 John Wiley & Sons, Ltd.
421
protein±protein interactions in real time. Nat. Struct. Biol. 7:
644±647.
Vijayakumar M, Wong KY, Schreiber G, Fersht AR, Szabo A, Zhou
HX. 1998. Electrostatic enhancement of diffusion-controlled
protein±protein association: comparison of theory and
experiment on barnase and barstar. J. Mol. Biol. 278: 1015±
1024.
Violette SM, Shakespeare WC, Bartlett C, Guan W, Smith JA,
Rickles RJ, Bohacek RS, Holt DA, Baron R, Sawyer TK. 2000. A
Src SH2 selective binding compound inhibits osteoclastmediated resorption. Chem. Biol. 7: 225±235.
von Kries JP, Winbeck G, Asbrand C, Schwarz-Romond T,
Sochnikova N, Dell'oro A, Behrens J, Birchmeier W. 2000.
Hot spots in beta-catenin for interactions with LEF-1,
conductin and APC. Nat. Struct. Biol. 7: 800±807.
Vu CB. 2000. Recent advances in the design and synthesis of SH2
inhibitors of Src Grb2 and ZAP-70. Curr. Med. Chem. 7: 1081±
1100.
Vu CB, Corpuz EG, Merry TJ, Pradeepan SG, Bartlett C, Bohacek
RS, Bot®eld MC, Eyermann CJ, Lynch BA, MacNeil IA, Ram
MK, van Schravendijk MR, Violette S, Sawyer TK. 1999.
Discovery of potent and selective SH2 inhibitors of the
tyrosine kinase ZAP-70. J. Med. Chem. 42: 4088±4098.
Walker CV, Caravatti G, Denholm AA, Egerton J, Faessler A, Furet
P et al. 2000. Structure-based design and synthesis of
phosphinate isosteres of phosphotyrosine for incorporation
in Grb2-SH2 domain inhibitors. Part 2. Bioorg. Med. Chem.
Lett. 10: 2343±2346.
Wall MA, Posner BA, Sprang SR. 1998. Structural basis of activity
and subunit recognition in G protein heterotrimers. Structure
6: 1169±1183.
Wallace LA, Dirr HW. 1999. Folding and assembly of dimeric
human glutathione transferase A1-1. Biochemistry 38:
16686±16694.
Way JC. 2000. Covalent modi®cation as a strategy to block
protein±protein interactions with small-molecule drugs. Curr.
Opin. Chem. Biol. 4: 40±46.
Wells JA. 1996. Binding in the growth hormone receptor
complex. Proc. Natl Acad. Sci. USA 93: 1±6.
Witter DJ, Famiglietti SJ, Cambier JC, Castelhano AL. 1998.
Design and synthesis of SH3 domain binding ligands:
modi®cations of the consensus sequence XPpXP. Bioorg.
Med. Chem. Lett. 8: 3137±3142.
Wlodawer A, Vondrasek J. 1998. Inhibitors of HIV-1 protease: a
major success of structure-assisted drug design. A. Rev.
Biophys. Biomol. Struct. 27: 249±284.
Xie ZH, Schendel S, Matsuyama S, Reed JC. 1998. Acidic pH
promotes dimerization of Bcl-2 family proteins. Biochemistry
37: 6410±6418.
Xie D, Gulnik S, Gustchina E, Yu B, Shao W, Qoron¯eh W, Nathan
A, Erickson JW. 1999. Drug resistance mutations can affect
dimer stability of HIV-1 protease at neutral pH. Protein Sci. 8:
1702±1707.
Xu D, Lin SL, Nussinov R. 1997a. Protein binding versus protein
folding: the role of hydrophilic bridges in protein associations. J. Mol. Biol. 265: 68±84.
Xu D, Tsai C-J, Nussinov R. 1997b. Hydrogen bonds and salt
bridges across protein±protein interfaces. Protein Engng 10:
999±1012.
Yang W, Rozamus LW, Narula S, Rollins CT, Yuan R, Andrade LJ,
Ram MK, Phillips TB, van Schravendijk MR, Dalgarno D,
Clackson T, Holt DA. 2000. Investigating protein±ligand
interactions with a mutant FKBP possessing a designed
speci®city pocket. J. Med. Chem. 43: 1135±1142.
Yanofsky SD, Baldwin DN, Butler JH, Holden FR, Jacobs JW,
Balasubramanian P, Chinn JP, Cwirla SE, Peters-Bhatt E,
Whitehorn EA., Tate EH, Akeson A, Bowlin TL, Dower WJ,
Barrett RW. 1996. High af®nity type 1 interleukin-1 receptor
antagonists discovered by screening recombinant peptide
libraries. Proc. Natl Acad. Sci. USA 93: 7381±7386.
Yao S, Brickner M, Paresmatos EI, Chmielewski J. 1998. Uncoiling
c-Jun coiled coils: Inhibitory effects of truncated fos peptides
on jun dimerization and DNA binding in vitro. Biopolymers
47: 277±283.
Yao ZJ, King CR, Cao T, Kelley J, Milne GWA, Voigt JH, Burke TR.
J. Mol. Recognit. 2002; 15: 405–422
422
A. V. VESELOVSKY ET AL.
1999. Potent inhibition of Grb2 SH2 domain binding by nonphosphate-containing ligands. J. Med. Chem. 42: 25±35.
Yeung D, Gill A, Maule CH, Davies RJ. 1995. Detection and
quanti®cation of biomolecular interactions with optical
biosensors. Trends Anal. Chem. 14: 49±56.
Yonehara M, Minami Y, Kawata Y, Nagai J, Yahara I. 1996. Heatinduced chaperone activity of HSP90. J. Biol. Chem. 271:
2641±2645.
Yudt MR, Koide S. 2001. Preventing estrogen receptor action with
dimer-interface peptides. Steroids 66: 549±558.
Zeden-Lutz G, Zuber E, Witz J, Van Regenmortel MH. 1997.
Thermodynamic analysis of antigen±antibody binding using
biosensor measurements at different temperatures. Anal.
Biochem. 246: 123±132.
Zeng J. 2000. Mini-review: computational structure-based design
of inhibitors that target protein surfaces. Combin. Chem.
High-Throughput Screen. 3: 355±362.
Zeng J, Fridman M, Maruta H, Treutlein HR, Simonson T. 1999.
Copyright # 2002 John Wiley & Sons, Ltd.
Protein±protein recognition: an experimental and computational study of the R89K mutation in Raf and its effect on Ras
binding. Protein Sci. 8: 50±64.
Zhang X, Morera S, Bates PA, Whitehead PC, Coffer AI,
Hainbucher K, Nash RA, Sternberg MJ, Lindahl T, Freemont
PS. 1998. Structure of an XRCC1 BRCT domain: a new
protein±protein interaction module. EMBO J. 17: 6404±6411.
Zutshi R, Chmielewski J. 2000. Targeting the dimerization
interface for irreversible inhibition of HIV-1 protease. Bioorg.
Med. Chem. Lett. 10: 1901±1903.
Zutshi R, Franciskovich J, Shultz M, Schweitzer B, Bishop P,
Wilson M, Chmielewski J. 1997. Targeting the dimerization
interface of HIV-1 protease: inhibition with cross-linked
interfacial peptides. J. Am. Chem. Soc. 119: 4841±4845.
Zutshi R, Brickner M, Chmielewski J. 1998. Inhibiting the
assembly of protein±protein interfaces. Curr. Opin. Chem.
Biol. 2: 62±65.
J. Mol. Recognit. 2002; 15: 405–422