Download in search of intramolecular chaperone

Protein Engineering vol.13 no.9 pp.617–627, 2000
Binding and folding: in search of intramolecular chaperone-like
building block fragments
Buyong Ma1, Chung-Jung Tsai2 and Ruth Nussinov2,3,4
1Laboratory
of Experimental and Computational Biology and 2Intramural
Research Support Program—SAIC, Laboratory of Experimental and
Computational Biology, NCI-FCRDC, Bldg 469, Rm 151, Frederick,
MD 21702, USA and 3Sackler Institute of Molecular Medicine, Department
of Human Genetics and Molecular Medicine, Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv 69978, Israel
4To
whom correspondence should be addressed, at the US address.
E-mail: [email protected]
We propose an intramolecular chaperone which catalyzes
folding and neither dissociates nor is cleaved. This
uncleaved foldase is an intramolecular chain-linked chaperone, which constitutes a critical building block of the
structure. Macroscopically, all molecular chaperones facilitate folding reactions and manifest similar energy landscapes. However, microscopically they differ. While
intermolecular chaperones catalyze folding by unfolding
misfolded conformations or prevent misfolding, the chainlinked cleaved (proregion) and uncleaved intramolecular
chaperone-like building blocks suggested here, catalyze
folding by binding to, stabilizing and increasing the populations of native conformations of adjacent building block
fragments. In both, the more stable the intramolecular
chaperone fragment region, the faster is the folding rate.
Hence, mechanistically, intramolecular chaperones and
chaperone-like segments are similar. Both play a dual role,
in folding and in protein function. However, while the
functional role of the proregions is inhibitory, necessitating
their cleavage, the function of the uncleaved intramolecular
chaperone-like building blocks does not require their subsequent removal. On the contrary, it requires that they
remain in the structure. This may lead to the difference in
the type of control they are under: proteins folding with
the assistance of the proregion have been shown to be
under kinetic control. It has been suggested that kinetically
controlled folding reactions, with the proregion catalyst
removed, lend longevity under harsh conditions. On the
other hand, proteins with uncleaved intramolecular chaperone-like building blocks, with their ‘foldases’ still
attached, are largely under thermodynamic control, consistent with the control observed in most protein folding
reactions. We propose that an uncleaved intramolecular
chaperone-like fragment occurs frequently in proteins. We
further propose that such proteins would be prone to
changing conditions and in particular, to mutations in this
critical building block region. We describe the features
qualifying it for its proposed chaperone-like role, compare
it with inter- and intramolecular chaperones and review
current literature in this light. We further propose a
mechanism showing how it lowers the barrier heights,
leading to faster folding reaction rates. Since these fragments constitute an intergal part of the protein structure, we
call these critical building blocks intramolecular, chaperonelike fragments, to clarify, distinguish and adhere to the
definition of the transiently associating chaperones. The
© Oxford University Press
new mechanism presented here differs from the concept of
‘folding nuclei’. While the concept of folding nuclei focuses
on a non-sequential distribution of the folding information
along the entire protein chain, the chaperone-like building
block fragments proposition focuses on a segmental distribution of the folding information. This segmental distribution controls the distributions of the populations throughout
the hierarchical folding processes.
Keywords: amino-terminus/chaperones/energy landscape/folding funnels/proregions/prosequences/protein binding/protein
folding
Introduction
The most important question in protein folding is how proteins
manage to find their native structures. The protein energy
landscape theory has provided an elegant way out of the longstanding baffling Levinthal paradox. However, it is still not
completely understood what is the driving force leading to the
native conformation and how proteins are able to overcome
barriers between local traps with similar thermodynamic
stabilities (Li et al., 1998). The hierarchical folding model
(Baldwin and Rose, 1999a,b) partially answers this question.
Evidence from folding intermediates and transition states
suggests that folding begins locally and that the formation of
native local structures precedes the formation of tertiary
interactions. However, it is still an open question how the
secondary structures reliably fold into tertiary structures. Here
we propose that in some cases, the missing step in the current
hierarchic folding model is that there may exist a segment in
the protein chains acting like an intramolecular chaperone, to
mediate and guide the final assembly of the protein tertiary
structure. To clarify the ensuing discussion, we note that
the ‘intramolecular chaperone-like’ segment described below
differs from the proregion. Unlike the proregion or the prosequence, which is covalently linked in the newly synthesized
polypeptide chain, catalyzes the folding reaction and is subsequently cleaved off to generate the mature protein, the
uncleaved intramolecular chaperone-like building block proposed here remains covalently attached and forms an integral
part of the protein for the duration of its lifetime. Hence, for
consistency with the prevailing definition (Ellis, 1997) and to
distinguish it from regular molecular chaperones, we use the
term chaperone-like.
Binding and folding are similar processes, governed by
similar principles (Tsai et al., 1998, 1999a). Consistently, a
chaperone can be either a separate molecule or chain-linked.
Both have been documented extensively. Different classes of
molecular chaperones exert their effect in different ways, either
by temporarily hosting the misfolded proteins in their cavities
or by binding to hydrophobic patches on their surfaces (Ellis,
1997, 1998; Netzer and Hartl, 1998; Ellis and Hartl, 1999).
Previously, it has been suggested that molecular chaperones
act through direct catalysis of the folding proceses. However,
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B.Ma, C.-J.Tsai and R.Nussinov
recently it has been shown that the action of the chaperonins
might be by preventing misfolding or by forcing unfolding of
a protein trapped in a misfolded conformation (Walter et al.,
1996; Shtilerman et al., 1999). These chaperones have no
effect on the final structure of a protein. In contrast, the chainlinked molecular chaperones have been proposed to act by
conveying steric information that is essential for correct folding
(Shinde et al., 1997). However, subsequently, the fragment
playing this role is cleaved off. Hence, in this sense, in all
three cases the chaperone molecules or the relevant fragments
act in a similar way, namely, via transient binding. Nevertheless, there is a difference between the molecular chaperones
and the fragment chaperones: in the first case, the molecular
chaperones are believed to aid through binding to intermediate
states, to prevent misfolding, with subsequent release. On the
other hand, in the intramolecular chaperone proregion, the profragment is attached to the polypeptide chain until the chain
achieves its final, native conformation. Only then it is selfcleaved.
Here we propose a class of intramolecular chaperone-like
fragments, those that are chain-linked and uncleaved. These,
permanently linked intramolecular fragment chaperones recur
frequently in proteins. While they have not been recognized
as such, perhaps owing to the fact that they remain attached
to the molecule throughout its lifetime, Shinde and Inouye
(1994) have searched for sequence similarity between motifs
they have identified in prosequences and other, related and
unrelated proteins, looking for potential significance. Here we
argue that such a permanently linked intramolecular chaperone
is a building block of the protein structure (Tsai et al., 1999a,b;
Kumar et al., 2000), is frequently (although not necessarily,
as discussed below) located at the amino-terminus of the
molecule and is essential for obtaining the native conformation.
If it is chopped off, the remainder of the molecule may still
attain a stable structure, but it will be non-native. This type
of (usually) amino-terminal fragment is in contact with other
building blocks of the structure, mediating their interactions.
In its absence, these building blocks are still likely to be in
their native conformations, but their association is non-native.
We further argue that this type of intramolecular chaperonelike building block is likely to be more frequently observed
in cases of non-sequential folding. In such cases, it serves to
mediate the interactions between the other building blocks,
preventing their potential non-native contacts. Such an aminoterminus building block fragment is then likely to be in contact
with a number of building blocks. For example, it may
associate with the carboxy-terminus building block and/or
concomitantly with other, internal blocks. For clarification,
Figure 1 presents a schematic drawing illustrating an uncleaved,
‘hidden’, intramolecular chaperone-like fragment. The figure
further illustrates the consequences of either its absence or of
its being flipped out and misfolded. Either way, under such
circumstances, stable, but non-native interactions between the
remaining building blocks take place. In contrast, if other
building blocks are absent, the population time of the remainder
of the native structure is still the highest among all (nonnative) alternative conformations.
With the goal of avoiding confusion in the terminology,
Ellis (1997) has recently provided two useful and simple
criteria qualifying a candidate molecule or fragment, as a
molecular chaperone: first, a molecular chaperone must assist
the non-covalent assembly or disassembly of other proteincontaining structures, with the mechanisms by which this role
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Fig. 1. A schematic illustration of the concept of uncleaved intramolecular
chaperone-like building block and the criteria for its selection. The protein
is dihydrofolate reductase. (a) The building block playing the role of the
chaperone is frequently at the amino-terminus. It should mediate the
interactions between the other building blocks of the structure. Here, this
building block is in red. The interactions involve knotted loop–loop
interaction (blocks 1 and 3) and side-chain hydrogen bonding interaction
between Asn18 and Ser49 (black dots between blocks 1 and 2; for side
chain, see Figure 3). (b) The candidate intramolecular chaperone-like
fragment is removed. The second (green) and third (blue) building blocks
are now in a hypothetical ‘open’ conformation. This type of situation may
lead to non-native interactions between the two building blocks. (c) The
non-native association which results is stable, constituting a meta-stable
state. If, however, the N-terminus is added to the solution, the conformation
will flip back to its native state, as observed in the studies of the proregion.
is carried out being irrelevant; second, it must not be a
component of these structures when they are carrying out their
normal functional roles. While our proposed building block
abides by the first criterion, it fails the second. Hence, adhering
to this definition, the uncleaved building block which guides
the folding of the molecule to which it is permanently attached
is not a molecular chaperone. Since, however, it bears a
resemblance to molecular chaperones, particularly the proregion, we distinguish between them by resorting to the term
intramolecular chaperone-like folding guide or building block.
Below, we first describe the intermolecular chaperones
and the intramolecular, proregion chaperone fragments which
undergo proteolysis after the molecule attains its native state.
Next we describe our proposed intramolecular, uncleaved
chaperone-like building blocks. We review current literature,
showing it to be consistent with our uncleaved intramolecular
chaperone-like building block proposition.
Intermolecular and intramolecular chaperones: the need
for guidance and catalysis in protein folding
Proteins are critical for practically all biological processes. To
ensure proper function, they need to have the ‘correct’ native
structure in the ‘right’ population, with the ‘proper’ redistribution of the conformations following the binding process.
Anfinsen (1973) had demonstrated that all the information
necessary for the folding of a protein into an active conformation is encoded in its amino acid sequence. However, it is
still unclear how nature has engineered the protein folding
mechanism. There are two questions in this regard: the first
relates to the dynamics, namely, how does a protein manage
to reach its native state, whereas the second concerns the
‘static’ aspect, namely, how is the structural information
distributed in the sequence? Regarding the first, dynamic,
property of protein folding, it is now clear that protein folding
does not always lead to the global free energy minimum and
that functional proteins are not necessarily in the global free
energy minimum state. Proteins can misfold and aggregate.
Intramolecular chaperone-like building blocks
On the other hand, there is increasing evidence that for some
proteins, kinetically selected states play functional roles. To
avoid misfolding and reach kinetically stable states, proteins
may need help from chaperones, particularly in vivo, where
the protein concentration is high.
Most of the chaperones known to date are distinct molecules
whose role is to prevent misfolding. Among these, two families
have been studied particularly well. The first is the chaperonins,
which include the GroEL, and the second is the smaller
chaperone proteins (Ellis, 1998; Netzer and Hartl, 1998; Ellis
and Hartl, 1999). The mechanism differs between these two
families. The chaperonins are large, multi-subunit allosteric
proteins, with central cavities into which the misfolded proteins
enter, with subsequent ejection (Shtilerman et al., 1999). On
the other hand, molecular chaperones belonging to the second
family appear to act via binding to the surface of misfolded
proteins, at sites with an extensive exposed non-polar area.
This second family consists of small proteins and hence cannot
have a cavity large enough to hold the misfolded proteins. Both
the chaperonins and most of the small molecule chaperones
apparently do not recognize specific sequences or well-defined
structures. This mostly sequence non-specificity in binding is
in contrast to the chain-linked, sequence- and structure-specific
intramolecular chaperones discussed below. Among the few
exceptions, cases such as that of the bacterial lipase limA
chaperone (Hobson et al., 1993) which is separated from the
lipase (lipA) by only three nucleotides and is lipase-specific,
can be considered as evolutionarily related to the intramolecular
proregion chaperones.
The third family is the cleaved-off intramolecular chaperones. This is the class that Ellis has aptly christianed ‘steric
chaperones’ in his recent insightful mini-review (Ellis, 1998).
It is well known that some proteins are synthesized with an
extra fragment at their amino termini. This fragment is essential
for correct folding (Baker et al., 1992). However, after the
protein folds, these fragments are degraded. Such cases typically occur in serine proteases, such as subtilisin (Figure 2a),
α-lytic protease (Figure 2b) and aqualysin from bacteria
and carboxypeptidase Y from yeast. These fragments act as
inhibitors, covering the active sites of the enzymes, hence it
is essential that they be cleaved and digested for the enzyme
to be functional. However, if the fragment is cleaved off in
the construct, prior to protein synthesis, the newly synthesized
chain misfolds. On the other hand, if the cleaved off fragment
is mixed with the remainder of the chain in solution, a correctly
folded protein is obtained. If the fragment is added to a
solution containing an already misfolded chain, the chain
converts to its native fold. Hence, as pointed out by Shinde
and Inouye (1994), there is a major difference between the
intermolecular and intramolecular proregion chaperones. In
the former case, in the absence of the chaperone, under proper
conditions, the protein molecule would still fold into its native
state, albeit with reduced efficiency. This, however, is not the
case for intramolecular proregion fragments. In their absence,
the protein will misfold.
The elegant mechanism of the intramolecular chaperone
leads us to wonder whether first, it is a rule that prosegments
have to be cleaved off to fulfil the role of intramolecular
chaperones; Second, could the question of their cleavage be
function dependent? Third, is it possible that there exist
uncleaved, ‘hidden’ intramolecular chaperone-like building
block folding guides in some proteins? In addressing these
questions, we encounter the static property of protein folding,
namely, how is the ‘structural’ information for protein folding
distributed in its sequence?
The kinetic and thermodynamic roles of proregions in
protein folding
Proteases are synthesized as inactive precursors or ‘zymogens’,
to prevent unwanted protein degradation and to allow spatial
and temporal regulation of their proteolytic activity. Upon
sorting or following appropriate compartmentalization, zymogens are converted to active enzymes, typically via limited
proteolysis and removal of an ‘activation segment’. The sizes
of the activation segments range from dipeptide units to
independently folding domains comprising more than 100
residues. A common form of the activation segment is an
N-terminal extension of the mature enzyme or ‘prosegment’,
that sterically blocks the active site and thereby prevents
binding of substrates. In addition to their inhibitory role,
prosegments are frequently important for the folding, stability,
and/or intracellular sorting of the zymogens (Khan and James,
1998; Khan et al. 1999).
The function of a prosegment is to ensure that the protease
functions at the right place and time. Often, a pro-region has
no role in folding; rather, the mature sequence already contains
most of the information required to specify its native threedimensional conformation (Price-Carter et al., 1996;
Veldhuizen et al., 1999). The folding-related roles of the
proregion may be classified into two types of cases: In the
first, the proregion acts as an intramolecular chaperone to
overcome kinetic barriers in folding. In the second, the
proregion acts as a thermodynamic stabilizer, through
uncleaved disulfide bonds.
Over the last few years, Agard and co-workers have made
a number of profound observations on proregion chaperones,
particularly on α-lytic protease. They have shown that the
stability of the enzyme derives not from thermodynamics, but
from its kinetics. The proregion is the means through which
this is achieved. The co-evolution of this region facilitates
folding into the native state, which is not the most stable one.
However, the barrier separating it from the global minimum
is high enough to keep the α-lytic protease in its native,
functionally active state (Baker et al., 1992, 1993; Cunningham
et al., 1999). Specifically, the studies of Sohl et al. (1998)
have indicated that the proregion catalyzes protease folding
by directly stabilizing the folding transition state. Anderson
et al. (1999) have investigated the precursor, the complexed
structure of the proregion with the mature α-lytic protease
enzyme and the isolated proregion. They have shown that
there are substantial similarities in the secondary structures of
the precursor and the complex, but they differ in their tertiary
structures and in particular in their stability. Correlation with
the proregion has indicated that it is fully folded and stabilizes
the structure of the enzyme. The rate of folding of the precursor
was shown to be biphasic, with the fast phase dependent on
the rate of the proregion folding. Hence the proregion folds
first, with subsequent catalysis of the folding of the protease
domain (Anderson et al., 1999). On the basis of these observations, Agard and co-workers have proposed a model in which
the major role of the proregion is to bind to the β-hairpin at
the C-terminal domain of the α-lytic protease, to form a fivestranded β-sheet and thereby to position the β-hairpin correctly,
leading to the proper folding of the α-lytic protease.
Similar conclusions have been reached following mutational
studies of the subtilisin BPN⬘ prodomain (Wang et al., 1998;
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B.Ma, C.-J.Tsai and R.Nussinov
Fig. 2. Stereo view of the building block cutting results for two cases where native folding requires the presence of a proregion. (a) Subtilisin; (b) α-lytic
protease. The structures are taken from the PDB (Bernstein et al., 1977). Each building block is in a different color. The N-terminus is in red. The cutting
results can also be viewed at our web site at http://protein3d.ncifcrf.gov/tsai/anatomy.html. Here the third cutting level is depicted.
Ruan et al., 1999). Three mutations were engineered in the
prodomain region, with none in contact with the subtilisin
itself. By sequentially introducing these stabilizing mutations
in the prodomain, the equilibrium for folding of the prodomain
shifted dramatically, from 97% unfolded to 65% folded. As
the prodomain was stabilized, the folding reaction of the
subtilisin became faster and distinctly biphasic. Furthermore,
consistently, the recent exciting work of Shinde et al. (1997,
1999) has elegantly illustrated that a mutation that has been
engineered in this 70-residue Pro fragment resulted in an
alternately folded subtilisin molecule, which has ‘memorized’
the altered proregion conformation. This finding again illustrates the importance of the conformation of Pro and that its
folding constitutes the first step in the folding of the precursor.
Proteins folding with the assistance of Pro have been shown
to be under kinetic control (Baker et al., 1992; Cunningham
et al., 1999). Sauter et al. (1998) provide a compelling
argument, rationalizing this choice by evolution. They point
out that since proteins utilizing Pro to fold function in proteaseinfested environments, the need to be resistant to proteases is
particularly acute. High kinetic barriers solve their problem.
During folding, Pro permits lowering of the barriers, leading
620
to the folded conformation. However, following the removal
of the catalyst, climbing the barrier presents a very difficult
hurdle, sustaining the native folds. Hence, in this case, the
folding reaction is practically irreversible.
Aside from this kinetic role of the Pro region, recently it
was demonstrated that a propeptide also acts to enhance the
thermodynamic stability of the mature protease. Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with
propeptides that remain attached to the active enzyme via a
disulfide bridge. It is likely that these propeptides are functionally important in the active enzymes, as well as in the
zymogens. Kardos et al. (1999) have investigated the role of
the disulfide-linked propeptide in the chymotrypsin(ogen) by
comparing the stabilities of the wild-type with those of mutant
proteins which lack propeptide–enzyme interactions both in
their zymogen (chymotrypsinogen) and in their active (chymotrypsin) forms. The mutants exhibited a substantially increased
sensitivity to heat denaturation and to guanidine hydrochloride
unfolding and a faster loss of activity at extremes of pH
relative to those of their wild-type counterparts. The guanidine
hydrochloride denaturation experiments indicated that the covalently linked propeptide provides about 24 kJ/mol of free
Intramolecular chaperone-like building blocks
energy of extra stabilization (∆∆G). Experimental evidence
has further suggested that the propeptide of chymotrypsin
restricts the relative mobility between the two domains of the
molecule (Kardos et al., 1999).
The important case of the chymotrypsinogen demonstrates
that the propeptide can have a thermodynamic role and, at the
same time, need not be cleaved off. Thus, it is reasonable to
argue that it is possible that there exists an uncleaved, ‘hidden’
intramolecular chaperone-like building block in some proteins.
Unlike in the case of the proregion, in our case here of the
uncleaved intramolecular chaperone-like catalyzed folding, the
reaction is likely to be largely controlled by thermodynamics.
This is the most frequently observed situation in proteins. We
will formulate the concept and support our proposal with
dihydrofolate reductase (DHFR).
All proteases have proregions: some fulfil both functional
and intramolecular chaperone roles and others, like the zymogens, do not catalyze the folding reaction. From the evolutionary standpoint, these two types of cases are similar: both are
synthesized as a single sequence, with the proregions linked
to the enzymes and both have the same, inhibitory, functional
role. However, for some enzymes belonging to the first type,
nature has taken advantage of the proregions already being
there, to assist in the folding, to reach a kinetically, otherwise
inaccessible state. Hence there is an inherent evolutionary
difference between the proregion intramolecular chaperones
and molecules whose sole role is to catalyze folding, like
the chaperonins and the small-molecule chaperones. Such
molecules have a priori arisen for their explicit role in folding.
Evolutionarily, the uncleaved intramolecular chaperone-like
building block folding guide proposed here falls into the
proregion category, namely, initially its role may have been
designated for function. However, while already there, it may
have evolved to assist optimally in the folding as well.
The concept and mechanism of the intramolecular,
uncleaved chaperone-like building block fragment
Here we propose that a building block, preferably at the aminoterminus, plays the role of an intramolecular ‘steric chaperone’.
In its absence, the chain misfolds. This proposition extends
the range of intramolecular chaperones. There are several
hypothetical reasons why evolution would choose to have a
chaperone that is chain-linked and ideally at the NH2 termini.
First, in vivo there is a high degree of crowding. Crowding
will enhance misfolding of polypeptide chains. A chaperone
molecule would help by preventing misfolding. A chaperone
in the form of a fragment that is chain-linked increases the
effective concentration of the chaperone and hence its efficiency. Second, if the fragment is linked at the amino-termini,
rather than at the carboxy-termini, it is more effective, as it
prevents misfolding a priori. These first two considerations
are fulfilled by the proregion (Ellis, 1998). However, in the
proregion case, the fragment guarding against misfolding is
cleaved off, whereas here we propose that the fragment remains
in the mature protein. From the evolutionary standpoint,
cleaving a building block fragment and digesting it are wasteful,
unless their role is inhibition. Otherwise, it is more efficient
if the fragment remains and is utilized to fulfil a dual role,
both in folding and in protein function, such as participating
in the binding of a ligand.
Our proposition is based on the building block folding
model (Tsai et al., 1999a,b). A building block is a contiguous
sequence fragment, with a variable size. It is a highly populated,
transient structural unit. The stability of a building block derives
from its intra-fragment interactions. While the conformation of
the building block that we observe in the native state of the
protein is likely to be the one adopted by the fragment in
solution, that is, it reflects the most populated conformation,
the building block may also twist or open up and in so doing
lose its intra-building block interactions. A building block
constitutes a local minimum among all potential fragments.
According to the building block folding model, via combinatorial assembly, the building blocks associate to form independently folding, compact, hydrophobic folding units. While the
conformations of most building blocks are preserved in the
folding process, the mutually stabilizing associations between
the building blocks may still result in alternative conformations
being selected in the combinatorial assembly process. The
building blocks folding model is a ‘practical’ model for
protein folding, postulating that folding is a hierarchic process
(Baldwin and Rose, 1999a,b). Here we propose that the aminoterminus which constitutes an intramolecular chaperone-like
fragment is such a building block. It need not be stable and
need not have a conformation which has a very high population
time. However, it constitutes a local minimum among all of
its neighbors and it has a higher population time than all other
alternative conformations. The formation of such a building
block during protein synthesis guards against misfolding, by
preventing the mis-association of other building blocks. In the
language of the energy landscapes and the folding funnels, a
building block intramolecular chaperone acts by lowering the
barrier of a misfolded conformation (Baker and Agard, 1994;
Gulukof and Wolynes, 1994; Dill and Chan, 1997; Karplus,
1997). Hence an intramolecular building block which acts as
a chaperone aids the trapped conformation climb out of its
minima-well. By being chain-linked, this goal is achieved
much more efficiently than otherwise.
Hence, specifically, here we argue that an intramolecular
chaperone does not work via binding to any intermediate
conformation of its adjoining building blocks and thereby
inducing it to undergo a conformational change to the native
conformation. On the contrary, here we propose that just as in
intermolecular binding in general, the binding of the aminoterminal intramolecular chaperone to its adjoining building
blocks is via conformational selection (Ma et al., 1999; Tsai
et al., 1999c; Kumar et al., 2000). Among all the available
conformations of the adjoining building blocks, those that bind
are the ones whose binding produces the most favorable
associations. And, in so doing, the equilibrium shifts in the
direction of the native building block conformations. Such a
proposition is consistent with the effect observed by Shinde
et al. (1997). The mutation they introduced does not cause a
direct, induced conformational change in the protease by
‘pushing’ and ‘pulling’ the protease into its ‘new native’ state.
Rather, the point mutation causes a change in the landscape,
reflecting the effect of the altered conformation of the aminoterminal building block fragment. The mutant building block
conformer preferentially selects different conformers from the
populations of conformers of its adjoining building blocks,
resulting in an altered subtilisin structure. However, after the
proregion containing the mutation is cleaved, with time,
we may expect the trapped altered structure to undergo
a conformational change, overcoming the barriers, to the
thermodynamically more stable native conformation.
For the sake of simplicity and clarity, here we outline the
three major considerations in selecting a candidate intramolecu621
B.Ma, C.-J.Tsai and R.Nussinov
lar chaperone-like building block fragment. They are highlighted in the schematic illustration in Figure 1. First, the
intramolecular chaperone fragment is most frequently at the
amino-terminus of the protein; second, the candidate intramolecular chaperone-like building block mediates the interactions
of other building blocks; and third, in its absence, non-native
interactions between the building blocks are likely to take
place. Figure 1a depicts a protein (dihydrofolate reductase)
cut into its three building block fragments. As the figure
shows, in this case, the N-terminal fragment (in red) mediates
the interactions of the second (green) and its sequentially
connected third (blue) building block. Figure 1b illustrates a
hypothetical conformation which may arise if the N-terminal
fragment is missing from the protein. Under such circumstances, a non-native association between the second and third
building blocks may take place. This association is stable, as
seen in Figure 1c, resulting in a meta-stable state. Such a
conformation has indeed been observed in simulations, in the
absence of the N-terminal fragment (Y.Sham, B.Ma, C.-J.Tsai
and R.Nussinov, unpublished results). Hence, as depicted in
these figures, the intramolecular building block chaperone-like
fragment is essential for achieving native interactions. Further,
the more stable the building block, the faster will be the
folding of the domain in which it resides.
Inspection of Figure 2a and b shows one likely reason for
the role played by the proregion in the folding reaction. In
both the α-lytic protease and subtilisin cases, the N-terminal
building block segment mediates the interactions of other
building blocks. It is essential that its conformation has a high
population time. If the native conformation of this building
block is present at a low concentration, the chances of nonnative interactions between the other building blocks will be
higher. Yet, inspection of Figure 2a shows that the conformation
of this building block fragment (in red), although constituting
a local minimum, is still relatively unstable. On the other
hand, its critical importance is seen from its mediating interactions with practically all other building blocks (in different
colors). We may expect that a sequentially connected proregion
would bind to this N-terminus fragment, since kinetically it is
most favorable and thereby stabilizes it, enhancing the presence
of this conformation. Consistently, a similarly unstable
N-terminal (red) building block situation is observed in the
α-lytic protease (Figure 2b).
Hence, in the subtilisin (Wang et al., 1998), the mutations
in the proregion shift the landscape of this domain, dramatically
increasing the population of the conformation which is most
favorable for binding with subtilisin and thereby driving the
folding reaction. The higher the stability of a region which is
in contact with other building blocks of the structure, the
higher its population time is expected to be and the folding of
the complex will also be faster. Wang et al. noted that under
these conditions, the rate is determined by the isomerization
of the trapped intermediate to the native complex structure.
Consistent with the building blocks hierarchical model, the
rate-limiting step here is the breaking of the non-native
contacts between the building blocks, in the trapped misfolded intermediate conformations. Hence, in both studies, the
critical step is a stable and fast folding proregion, which binds,
mediates and stabilizes less stable interactions between other
building blocks in these structures and in this way drives the
folding reaction.
Below we provide a concrete example showing this principle.
We utilize our recently developed procedure for dissecting
622
protein structures into building blocks, to produce progressively
their anatomy trees, in terms of protein folding (Tsai et al.,
2000).
Does DHFR contain an intramolecular, uncleaved
chaperone-like building block?
A few years ago, Matthews and co-workers (Gegg et al., 1997)
carried out a molecular dissection of the dihydrofolate reductase
(DHFR) of Escherichia coli. Their goal was to identify minimal
independent folding units. They generated eight overlapping
fragments, which span the entire 159 residues of the enzyme.
The generated fragments were analyzed using CD and fluorescence spectroscopy as a function of denaturation by urea. The
crystal structure of DHFR (solved with folate and NAD⫹;
Bernstein et al., 1977) is a doubly wound, parallel, α/β-sheet,
with four helices and eight strands. Visual inspection of the
structure illustrates the presence of two structural domains
(Figure 3). The first involves the adenine binding domain
(ABD), residues 37–88, and the second domain consists of the
two discontinuous fragments, the amino-terminus, residues 1–
36, and the carboxy terminus, residues 89–159. Spectroscopic
analysis of the eight fragments revealed that six out of the
eight, including the red fragment in Figure 3 (residues 1–36),
were disordered. Surprisingly, the ABD (in green) was also
found to be disordered. One fragment self-associated spontaneously and only the fragment consisting of residues 37–157
(in green and yellow in Figure 3) was found to possess
significant secondary and tertiary structure. The urea-induced
unfolding of this fragment showed a well-defined, two-state
process. The conformation of the fragment has non-native
contacts. Figure 3 represents the second level of the cutting
by our dissection algorithm (C.-J.Tsai and R.Nussinov, unpublished work). As the figure shows, three building blocks are
produced at this level, corresponding to the amino-terminal
fragment (in red), the ABD (in green) and the carboxy fragment
(in yellow). It is easy to see why in the absence of the red
fragment the protein misfolds. We propose that in the 37–159
fragment studied by Matthews and co-workers, the conformations of the green and yellow building blocks were in their
native state, but their association was non-native. The presence
of the red building block remedies this situation, by preventing
these green–yellow non-native contacts. Hence, in our terminology, the red building block serves as an intramolecular, aminoterminal, chain-linked chaperone-like fragment (see Figure 1).
The possible role of the chaperone-like N-terminus of the
dihydrofolate reductase is reinforced by the recent circular
permutation analysis of DHFR (Nakamura and Iwakura, 1999).
These experiments illustrated that the N-terminal region (up
to Met-16) play an important role in the overall tertiary fold,
rather than acting as a functional element. A homology study
of the DHFR indeed shows the high conservation of the
N-terminal segment. Superimposing five dihydrofolate reductase structures indicates a geometric conservation of this (red,
in Figure 4) fragment. As Figure 4 shows, the N-terminal
segment has much more overlap than other portions of these
proteins. This conservation can also be related to protein
function.
Given this rationale and the intriguing results of Shinde et al.
(1997, 1999), our proposition can be tested by experiments. If
valid, then first, mixing of the red and the green–yellow building
block fragments should yield the native state. Moreover, in
particular, such proteins may be more sensitive to mutations
in their intramolecular chaperone fragments. On the computa-
Intramolecular chaperone-like building blocks
Fig. 3. Stereo view of the cutting results for the dihydrofolate reductase. Here the second cutting level is shown. The building blocks are in different colors.
The N-terminus is in red. For further details, see caption to Figure 2.
Fig. 4. Superpositioning of five crystal structures of the dihydrofolate
reductase. The ribbon represents the dihydrofolate reductase from E.coli
PDB: 7dfr. The red ribbon highlights fragment of 1–36. The other, entirely
thin lines throughout the structures, are from PDB files 1dis, 1dyr, 1dls and
8dfr.
tional side, simulations of these fragments and of their generated mutants should also provide insight into the building
block fragment conformations and stabilities. For this system,
experiments attempting to mix the building block fragments
are restricted by the limited solubility of the 1–36 fragment
(Gegg et al., 1997). However, this problem is overcome by
protein fragment complementation studies. Murine dihydrofolate reductase has been divided into the N- and C-termini
fragments around residue 107. When fused to homodimerizing
GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners, active murine DHFR can be
reassembled (Pelletier et al., 1998, 1999).
Additional potential candidates for uncleaved intramolecular chaperones
As in the case of dihydrofolate reductase, there are many other
examples showing that a segment (mostly N-terminal) is
critical for folding of the entire protein.
ADP-glucose pyrophosphorylase is a key regulatory enzyme
in starch synthesis in most plant tissues. Laughlin et al. (1998)
deleted small N- and C-terminal peptides on the large and,
separately, on the small subunits from the potato protein and
co-expressed these with their wild-type subunit counterparts
from E.coli. Removal of the putative carboxy-terminal allosteric binding region from either subunit type prevents enzyme
formation, indicating that the carboxy terminus of each subunit
type is essential for proper subunit folding and/or enzyme
assembly as well as its suggested role in allosteric regulation.
Their results indicate that the N- and C-termini regions are
essential for the proper catalytic and allosteric regulatory
properties of the potato enzyme.
Recently, the L1 capsid protein of canine oral papillomavirus
has been used as an effective systemic vaccine that prevents
viral infections of the oral mucosa. The effect of this vaccine
is critically dependent upon the native L1 conformation. Chen
et al. (1998) generated a series of N- and C-terminal L1
deletion mutants. They found that (i) a deletion of the 26
C-terminal residues generated a mutant protein which folded
correctly both in the cytoplasm and in the nucleus, (ii) further
truncation of the L1 C terminus (67 amino acids) failed to
express the conformational epitopes and (iii) deletion of the
first 25 N-terminal amino acids also abolished expression of
the conformational epitopes. However, the native conformation
of this deletion mutant could be restored by the addition of
the human papillomavirus type 11 N terminus. These results
suggest that the L1 N-terminus has a critical role in protein
folding.
The β-subunit of Na,K-ATPase (βNK) is an additional
potential example. Complete truncation of the βNK N-terminus
allows for cell surface-expressed, functional Na,K-pumps that
exhibit, however, reduced apparent K⫹ and Na⫹ affinities, as
assessed by electrophysiological measurements. It has been
shown that the β N-terminus is not involved in the binding of
K⫹ and Na⫹ and the α-subunit. Rather, the effect of this
domain is through the correct folding of the whole β-subunit
(Hasler et al., 1998).
Most recently, Hayashi et al. (2000) studied the folding of
β-glucosidase. The enzyme is considered to be constructed
from four regions: the N-terminal catalytic domain, the non623
B.Ma, C.-J.Tsai and R.Nussinov
homologous region, the C-terminal unknown functional domain
and C-terminal residues. Based on gene manipulations, these
authors found that the folding information is unevenly distributed in these regions. The four types of truncated mutants at
the non-homologous region resulted in active enzymes with
slight modification of their characters. All four chimeric
enzymes shuffled at the C-terminal unknown functional domain
formed active enzymes. However, the two chimeric enzymes
shuffled at the N-terminal catalytic domain were obtained as
an inclusion body even though they were expressed with
GroEL/ES. Refolding of these proteins by slow dialysis was
not successful.
The above provide examples of either the N-terminal or
other segments in the protein which play an important role in
folding. These may constitute potential candidates for the
uncleaved intramolecular chaperone-like building blocks. Careful examination of their structures will be needed to verify (or
refute) them.
The separate chaperones and the uncleaved intramolecular
chaperone-like fragments
For the small chaperones, it currently appears as if a hydrophobic patch suffices for their binding, although the actual
mode of binding is still unclear. For the chaperonins, recent
work (Houry et al., 1999) suggests that β-sheets, preferably
with two hydrophobic cores on both sides, are the preferred
substrates. In agreement with these observations, Chen and
Sigler (1999) have shown that peptides adopting conformations
of a classical β-hairpin, a loop or an extended β-conformation
are good substrates for GroEL, with the β-hairpin binding
particularly tightly. Consistently, Chatellier et al. (1999) previously observed that an extended β-strand conformation binds
favorably to GroEL. Chatellier et al. further illustrated that
the spatial distribution of residues which are in contact with
GroEL is also compatible with an α-helix conformation. Chen
and Sigler suggested that a β-conformation is well suited for
the non-specific binding, typical of the GroEL. Such binding
may be attained via hydrophobic patches of the residue side
chains, in addition to the backbone and side chains of the
β-strands hydrogen bonding with the chaperonins. This type
of non-specific interactions has already been proposed as a
mechanism for non-specific poly-reactive antibodies to a broad
spectrum of antigens. In particular, the binding site and the
mode of the interactions are reminiscent of the MHC binding
to extended peptides with variable sequences (Fremont et al.,
1992; Madden et al., 1993). Additionally, the range of φ/ψ
values of β-conformations is larger in the Ramachandran
plots than those of α-helices. Hence, β-sheets and extended
β-segments are well suited to non-specificity, with the edge of
the β-sheet positioned against the binding site of the GroEL.
Sauter et al. (1998) have recently reported the structure of
the α-lytic protease complexed with the non-covalently bound
proregion (Pro). The structure shows that a C-shaped Pro
surrounds the C-terminal β-barrel domain of the protease.
However, the binding interface is arranged in such a way that
the C-terminus of Pro is not adjacent to the N-terminus of the
α-lytic protease. It therefore appears that when Pro is chainlinked to the enzyme, the binding orientation of α-lytic protease
and Pro may differ from the most favorable conformation
when the two molecular parts are unlinked. It is conceivable
that when chain-linked, Pro may bind to its sequentially
connected building block neighbor, forming a seed to which
adjacent building blocks bind. In particular, it is noteworthy
624
that when unbound, Pro is stable, with its conformation similar
to that observed in the complex (Sauter et al., 1998). That,
however, is not the case for subtilisin, where the cleaved Pro
is unstable (Wang et al., 1998). On the other hand, in both
α-lytic protease and the subtilisin cases, Pro is likely to aid in
forming the active site regions, positioning itself in a way so
as to enable the subsequent autocatalylic self-cleavage reaction.
Hence, taken together, this suggests that first, the cleaved,
complexed structure is likely to differ to some extent from the
structure of the chain-linked, prior to the cleavage event;
Second, the folding pathways of the chain-linked molecule
and of the protease without the Pro differ, as implied by the
cutting and rearrangement observed in the complexed structure.
Furthermore, in both the α-lytic protease and the subtilisin
cases, Pro serves as a template, to which adjacent building
blocks bind, via selection of their most favorable conformers.
Yet, after cutting, we observe a difference in the stability of
Pro in the α-lytic protease as compared with that of subtilisin.
In the subtilisin, Pro is stabilized through binding to its adjacent
building blocks in the enzyme. Thus, while it is unstable,
nevertheless the population of the native conformation of the
Pro region of subtilisin is higher than that of all alternative
conformations. This is consistent with the observations of
Wang et al. cited above, namely that the subtilisin Pro is
95% unfolded. However, following the mutations they have
introduced, Pro was observed to be 65% folded, with a marked
increase in the folding rate, explained by the higher population
time of Pro. Hence, the more stable the proregion, the faster
is the rate of folding. α-Lytic protease, with a stable Pro,
should fold faster than subtilisin. A similar observation has
recently been made for the dominant role of the prosegment
of prorenin in determining the rate of activation. Human
prorenin activation by acid or by trypsin is faster than that of
rat prorenin by two orders of magnitude. In an elegant
experiment, two chimeric mutant prorenins were produced.
The rate of activation of the first chimera, hPro/rRen, composed
of human prorenin and the rat active renin segment, was as
fast as the wild-type human prorenin. On the other hand, the
rate of activation of the second chimera, rPro/hRen, composed
of the rat prorenin prosegment and the human active renin
segment, was as slow as that of the wild-type rat prorenin.
These results suggest that the rate of activation of prorenin is
predominantly determined by the N-terminal prosequence
(Suzuki et al., 2000).
Consistently, in the case of the uncleaved intramolecular
chaperone-like proposed here, we may expect that the more
stable the building block fragment acting as the intramolecular
chaperone, the faster will be the rate of folding. The higher
the stability, the larger is the population of the building block
serving as a template for the binding of other building blocks
of the structure. A similar behavior has already been observed
in ‘simpler’, two-state proteins. Folding of acylphosphatase is
accelerated by the stabilization of local secondary structure
elements (Chiti et al., 1999), although these do not play
the role of intramolecular chaperones. Hence, going back to
the dihydrofolate reductase example, we may predict that if
the (red) N-terminal fragment is stabilized, e.g. via mutations,
the rate of folding will increase. This prediction may be tested
experimentally.
As for the kinetic control of protein folding, there are
additional cases which fold without a Pro region and their
folding still appears to be controlled by kinetics. Lomize et al.
(1999) have simulated the folding of four proteins, adhering
Intramolecular chaperone-like building blocks
to three criteria: burial of non-polar side chains, saturation of
‘hydrogen-bonding potential’ and stereochemical quality, as
reflected in close packing with no hindrance or holes. They
observed that some of the models they obtained had fewer
exposed non-polar side chains, more hydrogen bonds and
smaller holes than the corresponding crystal structures. This
implies that the native structures of such proteins are under
kinetic rather than thermodynamic control. In kinetically controlled folding reactions, with time, eventually the conformations will flip to their most stable state. However, the barriers
are such that the time-scales may not be biologically relevant.
To date, it has been shown that folding pathways can be
inferred from native structures (Alm and Baker, 1999a,b;
Galzitskaya and Finkelstein, 1999; Munioz and Eaton, 1999;
Tsai et al., 2000). An intriguing problem is how to infer which
pathways are thermodynamically and which are kinetically
controlled. Being able to distinguish between the two would
constitute an important advance in the protein folding problem.
To be very stable, a protein has a choice of two options: either
to have a substantial energy gap between the native and the
non-native conformations or to be surrounded by significant
barriers. Hence a related question is an understanding of the
general merit of kinetic versus thermodynamic control, where
applicable.
Some predictions
The above rationale leads us to make several predictions:
(i) If an N-terminal building block which appears to constitute
an intramolecular chaperone-like fragment is removed and
the structure is still unchanged, then that building block
does not play this proposed role. Conversely, if we add
an N-terminal building block and the global structure
changes, then we have created a new intramolecular
chaperone-like entity.
(ii) Mutations in the N-terminal intramolecular chaperonelike building block are likely to have a larger effect on
the global protein conformation, as compared with their
occurrence elsewhere in the structure. This is consistent
with the observation made by Shinde et al. (1997) on
‘protein memory’. The altered N-terminal building block
template conformation may select altered building block
conformers with which it associates, leading to a different
conformation.
(iii) Furthermore, sequence changes in an N-terminal chaperone-like building block may lead to entirely new folds.
It is well known that most mutations are tolerated, with
little, if any, effect on the protein structure. On the other
hand, some mutations have drastic effects, sometimes with
extreme consequences. If mutations cause a substantial
change in the conformation of the N-terminal building
block intramolecular chaperone-like fragment, such a
drastic structural alteration may ensue. This may occur
either via selection of altered building blocks conformers
or through an altered, new association between them (e.g.
as in Figure 1c). Either way, such a change may lead to
a new protein fold. Evolution and protein engineering
may take advantage of such a mechanism.
Conclusions
There are four major differences between the intermolecular
and intramolecular proregion chaperones. First, while the
intermolecular chaperones are sequence non-specific, the pro-
region is relatively sequence specific, accepting only a certain
range of mutations. Still, even then, the mutations which
change their own structure affect the conformation or the
folding rate of the protease whose folding they catalyze.
Second, the catalytic mechanism is different: while both work
by lowering the barrier heights, the intermolecular chaperones
or at least the chaperonins catalyze the folding reaction by
unfolding misfolded conformations. On the other hand, the
proregion catalyzes the reaction by directly assisting in the
folding, through binding and stabilizing adjacent native building block conformations and thereby increasing their populations. Hence, macroscopically, in both intermolecular and
intramolecular proregion chaperones, the funnel energy
lanscape is similar; However, the microscopics differ. Third,
there is a difference in the efficiency of the inter- as compared
with the intramolecular chaperones. As the latter are chainlinked, their effective local concentration is higher. Fourth, in
the case of the proregion, the corresponding proteins which
fold with their assistance are under kinetic rather than thermodynamic control, unlike the intermolecular chaperone-assisted
folding reactions. In addition, the two differ in their evolutionary aspect: whereas the proregions have primarily evolved to
act as inhibitors, as indicated from their zymogen counterparts
and have also assumed their folding role, the sole role of the
intermolecular chaperones is to assist in the folding.
Here we propose that there are intramolecular chaperonelike building block fragments which fulfil the chaperone role
and are not cleaved. The concept of intramolecular chaperones
applies to a broad range of cases, where these fragments both
assist in the folding reactions and, in addition, play a role in
protein function. Hence, like the proregions, they fulfil a dual
role. However, unlike the proregions, as their function is not
to inhibit, they are uncleaved. Therefore, here we propose that
cleavage of intramolecular chaperones is function-dependent.
Mechanistically, the uncleaved intramolecular chaperone-like
cases resemble the proregion-catalyzed reactions, lowering the
barrier heights by selectively binding to the most favorable
adjacent building block conformations. In this way, the equilbrium in the solution shifts toward these conformations, further
driving the folding reaction (Tsai et al., 1999b; Kumar et al.,
2000). These intramolecular chaperone-like folding guides
fulfil all the hallmarks of the intramolecular proregion-catalyzed reactions, with two differences: First, they remain
covalently linked to their parent molecule after the native state
has been reached; second, consequently, like the intermolecular
chaperones, the folding reactions that they assist are controlled
by thermodynamics. We suggest that this type of uncleaved
intramolecular chaperone-like catalyzed folding reactions are
a frequent occurrence. However, to preserve the definition of
the chaperones, they are termed chaperone-like.
The mechanism proposed here is entirely different from the
concept of ‘folding nuclei’. The concept of folding nuclei
focuses on a non-sequential distribution of the folding information which controls the initial step in the folding process along
the whole protein chain. On the other hand, our chaperone-like
building block fragments proposition focuses on a segmental
distribution of the folding information, which controls the
population distribution throughout the hierarchical folding
process.
Protein folding can be viewed as a process of intramolecular
recognition. Hence, protein folding and protein–protein association, which involves intermolecular recognition, are similar
processes (Tsai et al., 1998, 1999b). Both involve conforma625
B.Ma, C.-J.Tsai and R.Nussinov
tional selection, with subsequent population shifts in favor of
the depleted conformer and thereby continue the binding/
folding process. In both, the driving force is the hydrophobic
effect, although charge and polar complementarity are also
important contributors (Tsai et al., 1997; Xu et al., 1997).
Recently, Eisenberg and co-workers demonstrated the implications of this concept for the detection of protein function and
protein–protein interactions directly from genome sequences
(Marcotte et al., 1999). Here we illustrate its implications for
the molecular chaperones. The major difference is in the chain
linkage and hence in the efficiency.
Acknowledgements
We thank Drs Sandeep Kumar and Yuk Sham and, in particular, Jacob
V.Maizel for many helpful discussions. The research of R.Nussinov in Israel
has been supported in part by grant number 95-00208 from the BSF, by the
Center of Excellence administered by the Israel Academy of Sciences, by a
Magnet grant, by a Ministry of Science grant and by Tel Aviv University
Basic Research and Adams Brain Center grants. This project has been funded
in whole or in part with Federal funds from the National Cancer Institute,
National Institutes of Health, under contract number NO1-CO-56000. The
content of this publication does not necessarily reflect the view or policies of
the Department of Health and Human Services, nor does mention of trade
names, commercial products or organization imply endorsement by the US
Government.
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Received May 15, 2000; accepted July 14, 2000
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