Download Identification and Structural Characterization of the ATP/ADP

Cell, Vol. 90, 65–75, July 11, 1997, Copyright 1997 by Cell Press
Identification and Structural Characterization
of the ATP/ADP-Binding Site in the Hsp90
Molecular Chaperone
Chrisostomos Prodromou,* S. Mark Roe,*†
Ronan O’Brien,* John E. Ladbury,*
Peter W. Piper,* and Laurence H. Pearl*†
* Department of Biochemistry and Molecular Biology
† Joint UCL/LICR X-Ray Crystallography Laboratory
University College London
Gower Street
London WC1E 6BT
United Kingdom
Summary
Hsp90 molecular chaperones in eukaryotic cells play
essential roles in the folding and activation of a range
of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly
understood, and the involvement of ATP in particular
is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific
adenine nucleotide binding site homologous to the
ATP-binding site of DNA gyrase B. This site is the same
as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking
the binding of nucleotides to Hsp90 and not the binding
of incompletely folded client polypeptides as previously suggested. These results finally resolve the
question of the direct involvement of ATP in Hsp90
function.
Introduction
The 90 kDa heat shock protein family (Hsp90) are ubiquitous molecular chaperones with essential roles in stress
tolerance (Borkovich et al., 1989) and protein folding
(Freeman and Morimoto, 1996). In eukaryotes, the cytoplasmic Hsp90s act as specific chaperones for a wide
range of client proteins involved in signal transduction,
cell cycle regulation, and hormone responsiveness.
Specific clients in mammalian cells include tyrosine kinases such as pp60v-src (Opperman et al., 1981) and
Sevenless (Cutforth and Rubin, 1994); serine/threonine
kinases such as Wee1 (Aligue et al., 1994), c-Raf (Stancato et al., 1993), and Cdk4 (Dai et al., 1996); helixloop-helix transcription factors (Wilhelmson et al., 1990);
tumor suppressers such as Rb (Chen et al., 1996) and
p53 (Sepehrnia et al., 1996); and the cytoplasmic receptors for steroid hormones such as estrogen, progesterone, and glucocorticoid (Joab et al., 1984). The
involvement of several of these client proteins in cell
proliferation and tumor progression has prompted interest in Hsp90 as a target for antitumor drugs. One such
compound, geldanamycin, has been shown to interact
directly with Hsp90 (Whitesell and Cook, 1996) and
appears to promote the degradation of client proteins
before they are fully activated (Schneider et al., 1996).
Higher eukaryotes have a distinct form of Hsp90 (GRP94/
GP96 or endoplasmin) localized to the endoplasmic
reticulum, where it is involved in the assembly of immunoglobulins (Melnick et al., 1992) and other proteins
destined for secretion or surface presentation. GRP94/
GP96 is also involved in the loading of proteosomegenerated antigenic peptides onto nascent MHC Class
I molecules (Li and Srivastava, 1993).
Hsp90 alone can prevent protein aggregation and promote refolding in vitro (Weich et al., 1992), but in vivo it
is functionally associated in multiprotein complexes with
a range of accessory proteins. Initially, client proteins
are brought to Hsp90 in a complex involving the ATPase
chaperone Hsp70/DnaK and cochaperones Ydj1/DnaJ
(Kimura et al., 1993) and p48/Hip (Höhfeld et al., 1995),
which interact with Hsp90 via p60/Hop/Sti1 (Smith et
al., 1993). These accessory proteins are subsequently
replaced in the Hsp90–client complex by an immunophilin (FKBP52, Cyp-40, etc.), when the client protein is
a steroid receptor (Smith and Toft, 1993), or p50/CDC37
(Stepanova et al., 1996) when the client protein is a
protein kinase. p60/Hop/Sti1, p50/CDC37, and the
various immunophilins compete for binding to Hsp90
(Owens-Grillo et al., 1996) and are presumed to interact
with a common site. The final step of conformational
maturation requires the acidic p23 protein, whose binding to Hsp90 complexes appears to be ATP-dependant
(Johnson and Toft, 1994, 1995).
Binding and hydrolysis of ATP is a well-described
component of the molecular mechanisms of the Hsp70/
DnaK (Flynn et al., 1991) and the Hsp60/GroEL (Roseman et al., 1996) classes of molecular chaperones. However, the involvement of ATP in the mechanism of action
of Hsp90 has been controversial for several years. Observations of apparent ATPase (Nadeau et al., 1992,
1993) and autophosphorylation (Csermely and Kahn,
1991) activities may have been due to trace amounts of
the various protein kinases for which Hsp90 is the specific chaperone and/or contamination with the true
ATPase chaperone, Hsp70/DnaK, with which Hsp90 is
functionally associated (Czar et al., 1994). Consistent
with this, reports of ATPase and autophosphorylation
activities have been very variable between laboratories
and appear to depend on the protein source and the
degree of purification attained (Weich et al., 1993).
Other work has suggested that ATP triggers substantial
conformational change in Hsp90 (Csermely et al., 1993)
and that ATP binding converts Hsp90 to a conformational form required for binding of the p23 maturation
factor to Hsp90–client protein–immunophilin complexes
(Sullivan et al., 1997). However, a comparative study of
highly purified Hsp90 and Hsp70/DnaK (Jakob et al.,
1996) has clearly demonstrated the absence of a significant inherent ATPase activity in Hsp90 compared with
Hsp70/DnaK. The apparent inability of Hsp90 to bind
adenine nucleotides was also demonstrated by the failure to photoaffinity label Hsp90 with 8-azido-ATP, to
bind Hsp90 to an ATP–agarose affinity resin, or to observe enhancement of the fluorescence of MABA-ADP
(N-8-[4-N9-methylanthranylaminobutyl]-8-amino adenosine diphosphate) by Hsp90, all of which occur with
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Structure of ADP/ATP–Hsp90 N-Domain Complex
67
Hsp70/DnaK. Although the apparent involvement of ATP
in p23 binding remains unexplained, it has become
widely accepted that unlike Hsp70/DnaK and Hsp60/
GroEL, Hsp90 does not bind ATP.
Although the structure of the intact Hsp90 molecule
has not yet been determined, crystal structures of an
amino-terminal domain identified by limited proteolysis
have been determined for yeast (Prodromou et al., 1997)
and human (Stebbins et al., 1997) proteins. Consistent
with the high homology among all Hsp90 sequences
(69% identity, yeast to human) the tertiary structure of
these two domains is extremely similar, consisting of a
highly twisted eight-stranded b sheet covered on one
face by a helices. The quaternary structures of the human and yeast N-domains observed in the crystals are,
however, quite different. The yeast N-domain crystallizes as a dimer in which the C-terminal b strands of
the sheets in each monomer make an antiparallel interaction, generating a continuously hydrogen-bonded
16-stranded sheet in the dimer. This dimeric sheet folds
back on itself, forming a roughly cylindrical channel between the two monomers, whose size and shape suggest that it could function as a molecular clamp, capable
of accommodating 8–10 residues of polypeptide chain
in an extended conformation (Prodromou et al., 1997).
In contrast, the human N-domain crystallized as a monomer, the b strand that forms the dimer interface being
disordered, and the potential peptide-binding channel
was not observed (Stebbins et al., 1997). The outside
helical faces of the monomers in the yeast N-domain
dimer are formed by residues, many of which are very
highly conserved in all Hsp90-family sequences. At
the center of this helical face, a deep pocket penetrates
to the surface of the buried b sheet and is the binding
site for the antitumor agent geldanamycin in a complex
with the human N-domain (Stebbins et al., 1997). On the
basis of this complex, it has been postulated that this
pocket is a binding site for segments of polypeptide
chain from incompletely folded client proteins. Geldanamycin is therefore proposed to act as a competitive
inhibitor of client–protein binding.
In light of recent observations that the structure of the
Hsp90 N-domain has a similar topology to an N-terminal
ATP-binding domain of the bacterial type II topoisomerase, DNA gyrase (Dunbrack et al., 1997), the involvement of ADP/ATP in the function of Hsp90 must again
be reexamined. Sequence motifs comprising the ATPbinding sites within the N-terminal domain of DNA gyrase, and conserved in other type II topoisomerases,
are also conserved in Hsp90 sequences (Bergerat et al.,
1997), raising the possibility that these sequences might
also constitute an ATP-binding site in Hsp90. Here, we
report the high resolution crystal structures of specific
complexes of Mg21-ATP and of Mg2 1-ADP with the
N-domain (residues 1–220) of the yeast Hsp90 chaperone, providing definitive evidence for the involvement
of ATP binding in the molecular mechanism of Hsp90.
Results and Discussion
Cocrystals of yeast Hsp90 N-domain and ADP, ATP, or
ATPgS were grown under conditions previously described for native crystals (Prodromou et al., 1996), but
with the addition of 5 mM nucleotide and 5 mM Mg21.
Electron density maps for Hsp90 complexed with Mg21ATP, Mg21-ADP, and Mg21-ATPgS were obtained at 1.8,
2.0, and 2.5 Å resolution, respectively.
Location of the ATP/ADP-Binding Site
Difference Fourier maps showed clear positive features
corresponding to the bound nucleotides, lying in a deep
pocket on the helical face of the N-domain (Figures 1a
and 1b). This pocket is bounded by the helices from
28–50 and from 85–94 on two sides, and the end of the
helix and loop from 117–124 and the loop from 81–85
on the other two sides. The base of the pocket is formed
by residues Ile-77, Asp-79, Val-136, Ser-138, Thr-171,
and Ile-173, whose side chains project up from the buried face of the b sheet. The electron density for the base,
sugar, and a phosphate groups, which make extensive
contacts within the pocket, is extremely clear in all the
complexes (Figure 1c). The electron density for the b
phosphate, which lies higher up in the pocket and makes
fewer contacts, is somewhat weaker, but no significant
electron density is present for the g phosphate in complexes with ATP or with the nonhydrolyzable analog,
ATPgS. This suggests that the phosphate has not been
lost by hydrolysis of ATP over the time scale of the
crystallization experiment but is truly disordered in the
crystals. Analysis of the binding of ATP and ADP to
the Hsp90 N-domain in solution by isothermal titration
calorimetry indicates dissociation constants of 132 6
47 and 29 6 3 mM, respectively, with binding stoichiometries close to 1 nt per N-domain monomer.
Nucleotide–Protein Interactions
The bound nucleotides make extensive interactions with
the protein and bound solvent in the pocket (Figure 2).
The adenine base penetrates into the pocket, making
only a single direct hydrogen bond to the protein, from
the exocyclic N6 amino group of the adenine base to
the carboxyl side chain of Asp-79 at the bottom of the
pocket. All the other hydrogen-bonding possibilities of
the adenine base are fulfilled by water molecules bound
by protein groups within the pocket. Thus, the second
hydrogen bond to adenine N6 is made by a water molecule bound by the peptide carbonyl of Leu-34; adenine
N1 is hydrogen bonded to a water molecule bound by
Figure 1. Nucleotide Binding by the Hsp90 N-Terminal Domain
(a) Secondary structure cartoon of the yeast Hsp90 N-domain dimer showing the position of the bound ADP/ATP. The base, sugar, and
phosphates of the bound nucleotide are colored green, red, and magenta, respectively.
(b) Stereo view of the ADP/ATP-binding site in one monomer.
(c) Stereo view of electron density for the bound nucleotide and associated solvent from the Hsp90 N-domain–ATP complex. The electron
density is from an Fo-Fc Fourier, phased from protein coordinates only, refined against the data at 2.0 Å using simulated annealing. Contours
are at 3.0s.
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Figure 2. ADP/ATP Interactions in the Nucleotide Binding Pocket of Hsp90
(a) Overall view of ADP bound in the pocket on the helical face of the Hsp90 N-domain monomer. The solvent accessible protein surface is
colored to reflect the electrostatic potential, going from negative (red) to positive (blue). The bound ADP molecule is colored as in Figure 1a.
(b) Schematic diagram of ADP interactions. Hydrogen bonds are shown as dashed lines, van der Waals interactions are indicated by fur.
(c) Stereo view of ADP bound in the nucleotide-binding pocket. Residues from the protein are drawn as green sticks, and the ADP is shown
in ball-and-stick representation with CPK colors for the atoms. Water molecules are red spheres, and the Mg2 1 ion is a white sphere. Hydrogen
bonds are shown as broken yellow rods, and the magnesium-ligand interactions as broken blue rods.
the side chains of Asp-79 and Thr-171 and the peptide
nitrogen of Gly-83; N3 of the adenine and O29 of the
ribose to a water molecule bound by Asn-92; and N7 of
the adenine to a water bound by the side chain of Asn37. One hydrophobic face of the adenine ring is in van
der Waals contact with the side chain of Met-84, but
the other face is effectively exposed to solvent.
O29 of the ribose also makes a direct hydrogen bond
to the side chain of Asn-92. Closer to the top of the
pocket, the a phosphate group makes hydrogen bonds
with the side chain of Asn-37 and the peptide nitrogen
of Phe-124. The b phosphate group makes an ion-pair–
hydrogen-bonding interaction with the side chain of Lys98 and interacts with several solvent molecules bound at
the mouth of the pocket. One possible solvent molecule
interacts with oxygens of the a and b phosphates, the
side-chain amide oxygen of Asn-37, and three other
solvent molecules in an approximately octahedral coordination and is tentatively identified as an Mg2 1 ion. No
binding of ADP or ATP to Hsp90 N-domain is observed
in calorimetric studies in the absence of Mg21. Crystals
grown in the presence of Mg21 or Mn21 alone do not
show bound metal ions, suggesting that the N-domain
has no inherent Mg21-binding site, but binds Mg2 1-ADP/
ATP. Other than the ordering of the side chain of Lys-98,
there are no significant changes observed between the
structure of the protein in the ADP or ATP complexes
and the free protein.
Structure of ADP/ATP–Hsp90 N-Domain Complex
69
Our observation of specific ADP/ATP binding to
Hsp90 completely contradicts the careful and widely
accepted biochemical analysis of Jakob et al. (1996),
who demonstrated clearly that Hsp90 could not be
specifically photoaffinity labeled with 8-azido-ATP, was
not retained on C8-ATP-agarose, and did not enhance
the fluorescence of MABA-ADP. In contrast, all these
reagents gave positive results with Hsp70/DnaK, leading
to the reasonable conclusion that Hsp90 does not bind
adenine nucleotides. We can understand these negative
observations by consideration of the different conformation of the adenine nucleotides bound to the two
different chaperones. In an Hsc70–ADP complex (Flaherty et al., 1994), the bound nucleotide adopts a fully
extended conformation with a 29-endo sugar pucker and
the g torsion angle in the ap conformation (for conformational definitions, see Moodie and Thornton, 1993). In
this conformation, the 8 position of the adenine is unhindered, and 8-substituted analogs would have little
difficulty binding to Hsc70. The nucleotide in the Hsp90–
ATP/ADP complex, however, has a much more compacted conformation, with a 39-endo sugar pucker and
a 1sc g torsion angle. In this conformation, the 8 position
is substantially hindered by the 5 carbon and a phosphate, so that this conformation cannot be adopted by
8-substituted ADP/ATP analogs (Figure 3). We suggest
that the negative results for nucleotide binding by Hsp90
obtained by Jakob et al. (1996) result from their use of
C8 adenine–substituted reagents, which are not able to
adopt the idiosyncratic conformation required for ATP/
ADP binding to Hsp90.
Implications for Geldanamycin Binding
The ATP/ADP-binding site we have identified has previously been shown to be the binding site for the antitumor agent geldanamycin on the N-terminal domain of
the human Hsp90 (Stebbins et al., 1997). Geldanamycin
consists of an ansa ring closed by an embedded benzoquinone, with a pendant carbamate group approximately halfway around the ansa ring. On the basis of
the interactions observed between geldanamycin and
Hsp90, Stebbins et al. (1997) suggest that the ansa ring
of geldanamycin imitates a pentapeptide in a turn conformation and therefore propose that the biological role
of the geldanamycin-binding site is in binding segments
of polypeptide chain from incompletely folded client
proteins. Our results clearly contradict this suggestion
and rather indicate that geldanamycin is acting as an
ADP/ATP mimetic, specific to the idiosyncratic set of
interactions offered by the nucleotide-binding site of
Hsp90. In support of this idea, we note that almost all
of the polar interactions described between geldanamycin and human Hsp90 have precise equivalents in
the specific interactions between yeast Hsp90 and
ADP/ATP (compare Figure 2c of this paper with Figure
6a in Stebbins et al., 1997). Most significantly, at the
bottom of the pocket, the direct hydrogen bond observed between the carbamate nitrogen of geldanamycin and the carboxyl side chain of Asp-93 in human
Hsp90 corresponds to the direct hydrogen bond between the adenine N6 and Asp-79 in yeast Hsp90. In
addition, the hydrogen bond between the carbamate
Figure 3. Comparison of Bound Nucleotide Conformations in Hsc70
and Hsp90
Conformations of ADP bound to (a) Hsc70 (Flaherty et al., 1994) and
(b) Hsp90. The N1, N6, which make specific contacts in the ADP/
ATP-binding pocket, are indicated, as is the adenine base C8 atom,
which is unhindered in the Hsc70-bound conformation but hindered
in the Hsp90-bound conformation.
oxygen of geldanamycin and the buried water bound
by Asp-93, Gly-97, and Thr-184 in human Hsp90 corresponds to the interaction between the adenine N1 and
the buried water bound by Asp-79, Gly-83, and Thr-171
in yeast Hsp90. Further up the pocket, the hydrogen
bond observed between the amide carbonyl of geldanamycin and the main-chain nitrogen of Phe-138 in human
Hsp90 corresponds to the hydrogen bond between an
oxygen of the a phosphate of ADP/ATP and the mainchain nitrogen of Phe-124 in yeast Hsp90. Similarly, the
hydrogen bond from the e amino of Lys-112 to a geldanamycin benzoquinone oxygen corresponds to the hydrogen bond/ion pair between Lys-98 and the b phosphate
of ADP/ATP. The hydrogen bonds made by the e amino
of Lys-58 to methoxy and carbonyl oxygens on geldanamycin have no direct equivalent in the yeast ADP/ATP
complex but may correspond to interactions between
the side chain of Lys-44 in yeast Hsp90 and solvent
molecules bound to the O29 and O39 oxygens of the
ribose sugar in ADP/ATP. The recognition that geldanamycin imitates the binding of ADP/ATP to Hsp90, rather
than peptides as previously suggested (Stebbins et al.,
1997), will be of considerable value in the further development of this and other compounds as antichaperone
agents, with potential applications in the treatment of
many cancers.
Structural and Functional Similarity of Hsp90
and DNA Gyrase B N-Terminal Domains
The overall tertiary fold of the yeast and human Hsp90
N-domains has a remarkable and totally unsuspected
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70
similarity to the N-terminal ATP-binding fragment of the
bacterial type II topoisomerase, DNA gyrase B protein
(Wigley et al., 1991). This similarity was not initially recognized by the authors of either the human or yeast
structures but was determined during the CASP2 structure-prediction competition (Dunbrack et al., 1997), to
which the yeast Hsp90 N-domain was submitted.
Optimal structural alignment of the N-terminal domains of yeast Hsp90 N-domain and the gyrase B using
the SSAP algorithm (Orengo and Taylor, 1996) brings
six b strands and five helices in the Hsp90 structure
into equivalence with an rmsd between 79 common Ca
positions of approximately 4 Å and almost superimposes the bound nucleotides from the two structures
(Figure 4). This degree of structural homology, taken
together with a clear functional similarity, would argue
strongly that both the Hsp90 and DNA gyrase adenine
nucleotide-binding domains are evolved from a common ancestor. However, even when aligned on the basis
of this structural equivalence (Figure 5), the amino acid
sequences of the two proteins only have around 10%
identity, suggesting that they diverged early in evolution.
While the central strands of the b sheet are very similar
in both Hsp90 and gyrase B, the arrangement of the
terminal strands is different. In the gyrase B N-domain,
the amino-terminal sequence from 2–15 is detached
from the body of the protein and participates in a dimer
interaction with a second molecule, whereas the equivalent sequence in Hsp90 forms the amino-terminal
Figure 4. Comparison of the ATP-Binding Domains of DNA Gyrase
B and Hsp90
(a and b) Side-by-side comparison of the backbone folds of Hsp90
(right) and DNA gyrase B (left) N-terminal domains. The amino-terminal strand in gyrase B and the C-terminal strand in Hsp90, which
participate in dimer formation, are highlighted in cyan and red, respectively. The lid segment is highlighted in magenta. Bound nucleotides are shown as green CPK models.
strand of the sheet. Conversely, in Hsp90 the C-terminal
strand from 205–220 extends from the body of the protein, making a dimeric interaction with a second molecule, whereas the equivalent sequence in gyrase B folds
back to form the C-terminal strand of the sheet.
The most significant difference between the structures of the Hsp90 and gyrase B N-domains is the conformation of the polypeptide sequence from residues
94–124 in Hsp90 and the corresponding sequence from
95–119 in gyrase B. This segment in gyrase B is an
extended loop of irregular conformation, folded down
onto the ATP-binding site as a lid, making contact with
the base and phosphates of the bound ATP. In Hsp90,
this segment consists of a short a helix, a loop, and a
short 310 helix and is packed against the helix and loop
formed by residues 10–27, away from the ADP/ATPbinding site. The difference in orientation of this otherwise topologically equivalent segment corresponds to
a hinge motion, pivoting at glycines 100 and 118 in
Hsp90 and at glycines 101 and 113 in gyrase B. This
segment is conformationally flexible in gyrase B and is
displaced from its closed conformation over the ATPbinding pocket in complexes with coumarin and cyclothialidine antibiotics and becomes disordered (Lewis et
al., 1996). While this segment is orientated away from the
ADP/ATP-binding site in both yeast and human Hsp90
structures, the local structure around the pivot residue
Gly-114 in human Hsp90 (Gly-100 in yeast) displays different conformations in different crystal forms, suggesting that the mobility seen in gyrase might also be
present in Hsp90. If this is the case, this segment in
Hsp90 might also be able to act as a lid closing over
the ADP/ATP-binding site and interacting with the nucleotide. In the crystals of yeast Hsp90, residues in this
segment are involved in crystal contacts that would significantly stabilize the open conformation for the lid,
despite the presence of nucleotides. From the present
data, the possibility of a dynamic variation of the Hsp90
lid conformation as a result of nucleotide binding is
speculative. Indeed, the extensive hydrophobic interface between the lid segment and the helix and loop
from residues 10–27 in Hsp90 suggests that this open
conformation is rather stable. The difference in conformation between the lids in gyrase B and Hsp90 may
actually be the result of selection of a different static
conformation in the evolution of Hsp90.
Consistent with the structural homology between
Hsp90 and DNA gyrase N-terminal domains, the conformation of ADP/ATP in the binding sites of Hsp90 and
DNA gyrase is remarkably similar, and many of the protein residues interacting with the nucleotides are conserved between the two proteins, constituting distinct
sequence motifs characteristic of type II topoisomerases and Hsp90s (Bergerat et al., 1997). In particular,
Asp-79, Gly-83, and Thr-171, which provide the direct
and solvent-linked interactions with the N1 and N6
atoms of adenine in Hsp90, correspond to Asp-73,
Gly-75, and Thr-165, which perform the same function
in gyrase B. A magnesium ion is also present in the
Hsp90-ADP/ATP-binding site in essentially the same
position in gyrase B. This ion contacts the a and b phosphates in the ADP and ATP complexes and the sidechain amide oxygen of an asparagine (37 in Hsp90, 46
Structure of ADP/ATP–Hsp90 N-Domain Complex
71
Figure 5. Structural Alignment of the Amino
Acid Sequences of Hsp90 and DNA Gyrase B
Alignment of the amino acid sequences of E.
coli DNA gyrase B (SwissProt: GYRB_ECOLI)
and S. cerevisiae Hsp90 (SwissProt:
HS82_YEAST) N-domains, based on the
alignment of their secondary structures by
the SSAP algorithm (Orengo and Taylor,
1996). Helices are shown as cylinders, with a
helices colored red, and helices with a primarily 310 conformation colored magenta. b
strands are shown as green arrows. Sequence identities are indicated by vertical
bars between the sequences, and the three
motifs identified as common to type II topoisomerases and Hsp90s (Bergerat et al., 1997)
are highlighted in red.
in gyrase B) conserved in both protein families (Bergerat
et al., 1997). There are some small differences between
the phosphate interactions made by Hsp90 and gyrase
B. For example, the b phosphate forms an ion-pair–
hydrogen-bonding interaction with the side chain of
Lys-98, which is conserved in Hsp90s but is Ala-100 in
gyrase. The major difference between the well-ordered
g phosphate in gyrase B and the disordered g phosphate
in Hsp90 may result from the open-lid conformation in
Hsp90, compared with the closed-lid conformation
found in gyrase B. However, interaction with the g phosphate coming from residues in the following domain may
also contribute to the ordering of this group in gyrase B.
A Role for ADP/ATP Binding in the
Mechanism of Hsp90
The results we present here unequivocally demonstrate
the existence of a specific ADP/ATP-binding site conserved throughout eukaryotic and bacterial Hsp90s,
necessitating a substantial reappraisal of much of the
current data relating to the biochemistry of Hsp90. The
ADP/ATP-binding site we have identified coincides
structurally with the binding site for geldanamycin identified by Stebbins et al. (1996) and therefore clearly corresponds to the ADP/ATP-binding site postulated by
Toft and his colleagues (Johnson and Toft, 1994, 1995),
which appears to regulate the binding of the maturation
factor p23 to Hsp90–client–immunophilin complexes.
Binding of p23 to Hsp90 is promoted by ATP or ATPgS
but inhibited by ADP (Sullivan et al., 1997) and thus
appears to be dependant on the presence of the g
phosphate. In the Hsp90–ATP complex, the disordered
g phosphate will be exposed at the surface of the
N-domain and could either interact with p23 directly,
forming part of a p23-binding surface on the N-domain,
or could interact with other parts of the Hsp90 molecule
to stabilize a conformation that favors p23 binding elsewhere. The nucleotide pocket lies near the middle of an
elongated channel that traverses the helical face of the
N-domain, which might provide part of an extended
binding site for p23. If the conformation of the lid in
Hsp90 is indeed variable, then a closed conformation
similar to that in DNA gyrase might be favored by interaction with the g phosphate of a bound ATP. This would
unmask a potential protein-binding surface formed by
the residues buried by the lid in its open conformation.
Interestingly, temperature-sensitive mutants of yeast
Hsp90 (Nathan and Lindquist, 1995) that cannot be simply attributed to destabilization of the protein core or
disruption of the nucleotide-binding site map in this loop
(Thr-22→Ile) or at the hinge of the lid (Thr-101→Ile).
In both of the two well-characterized ATP-dependant
chaperone systems, Hsp70/Hsc70/DnaK and Hsp60/
GroEL, binding and hydrolysis of ATP, and release of
ADP, are used to drive conformational changes that
cycle the chaperones through high affinity and low affinity states for incompletely folded protein substrates
(Roseman et al., 1996; Zhu et al., 1996). Given that we
have unequivocally demonstrated the existence of an
ATP-binding site in Hsp90, it is reasonable to speculate
whether Hsp90 operates by a similar mechanism. The
ATP affinity of the Hsp90 N-domain (≈132 mM) is sufficiently high as to guarantee that the ATP-binding site
will be fully occupied at cellular ATP concentrations.
Conversely, the affinity for ADP (29 mM), although higher
than for ATP, is probably insufficient to saturate the
binding site with ADP at cellular concentrations. At least
in terms of nucleotide affinity, therefore, Hsp90 is set
up to go through an ATP-binding–ATP-hydrolysis–ADPrelease cycle comparable to those of Hsp60/GroEL or
Hsp70/DnaK.
The binding of ATP by Hsp90 has been clearly demonstrated here, but the existence of an inherent ATPase
activity remains unresolved. The ATP-binding site of
Hsp90 is very similar to that of the proven ATPase DNA
gyrase B, and the catalytic glutamate (Glu-42) responsible for the ATPase activity of gyrase B (Jackson and
Maxwell, 1993) is conserved in the N-domain of Hsp90
(Glu-33), suggesting that Hsp90 is at least equipped for
an ATPase activity with essentially the same catalytic
mechanism as DNA gyrase B.
In isolation, the 24 kDa N-domain of gyrase B will
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neither bind nor hydrolyze ATP (Gilbert and Maxwell,
1994), and productive binding of ATP by DNA gyrase B
is dependent on interactions with the g phosphate from
residues in the following domain, which may provide a
means for coupling ATP hydrolysis to changes in the
relative juxtaposition of these domains (Wigley et al.,
1991). Although the isolated N-domain of Hsp90 does
bind ATP weakly, the g phosphate is disordered, and
an ATPase activity in the intact Hsp90 may also depend
on interactions to stabilize the conformation of the disordered g phosphate. As in DNA gyrase B, these interactions might be provided by residues in other domains,
giving a mechanism for ATPase-coupled conformational
changes, or might be provided by a separate protein,
such as p23.
Conformational changes of Hsp90 on addition of ATP
have been reported (Csermely et al., 1993), but the nature of these is unknown. The molecular-clamp structure
formed by the yeast Hsp90 N-domain dimer (Prodromou
et al., 1997) displays a conformational flexibility between
a closed form in which the loops at residues 160–168
from each monomer are in contact and an open form
in which they are separated by z8 Å (Figure 6). The
structure of the Hsp90 N-domain dimer is such that the
C-terminal strands forming the dimer interface swap
over topologically. This gives rise to the possibility that
regions of the structure beyond the C-terminus of the
N-domain from one monomer could interact with the
ATP-binding site of the N-domain of the other monomer
in the intact Hsp90 dimer. Such interactions either direct
or mediated by p23 would be very likely to influence the
relative juxtaposition of the N-domain with respect to
other parts of Hsp90 and consequently affect the conformation of the clamp (Figure 6c). Whether ATP binding
would serve to open the clamp or close it can only be
speculated upon at this stage. The situation is further
complicated by the presence of two equivalent ATPbinding sites in the dimer, allowing for ATP2 -, ADP2-, or
ADP1ATP-loaded states, with the possibility of positive
or negative cooperativity between them.
At present, we do not know what function this clamp
serves in the molecular mechanism of Hsp90. The channel defined by the clamp in the closed conformation
(Prodromou et al., 1997) is of the right shape and size
to accommodate a peptide chain in an extended conformation, making contact with 8–10 amino acids. The
clamp might provide the common binding site suggested for the various accessory factors, such as p60/
Hop/Sti1, p50/CDC37, and immunophilins, which need
to be exchanged at various stages of Hsp90-dependant
protein folding and activation. Alternatively, the clamp
might provide a site for binding parts of client proteins
during folding, i.e., binding and releasing segments of
polypeptide chain in response to ATP binding and hydrolysis, in an analogous manner to the Hsp70 chaperone.
The clamp might also be involved in an emerging role
for the endoplasmic reticulum Hsp90, GRP94/GP96, in
loading antigenic peptides, imported into the ER lumen
by the TAP transporter, onto MHC Class I molecules
(Li and Srivastava, 1993). Thus, immunodominant viral
peptides eluted from MHC-I on the surface of cells infected with vesicular stomatitis virus, are also elutable
from the GRP94/GP96 from the ER of the same cells
(Nieland et al., 1996). The size of the binding site offered
by the Hsp90 N-domain clamp conforms to the predominant length of antigenic peptides presented by MHC-I,
and the loading process appears to be ATP dependent
(Li and Srivastava, 1993). The high degree of conservation of the N-domain clamp between Hsp90s of the
cytoplasm and endoplasmic reticulum, suggest that the
cytoplasmic Hsp90 might have an analogous role in
transporting antigenic peptides from the proteosome to
the cytoplasmic face of the TAP transporter.
The data we present here finally resolves the controversy of ATP involvement in the function of Hsp90 and
correctly defines the action of the antitumor agent geldanamycin as an Hsp90-specific ATP mimetic. The possibility of ATPase activity inherent in Hsp90 is still not
Figure 6. A Model for the ATP-Dependent Conformational Changes
in the Molecular Clamp of the Hsp90 N-Terminal Domain
(a and b) Conformational flexibility of the N-domain dimer, in the (a)
closed and (b) open conformations (Prodromou et al., 1997). The
bound nucleotides observed in the closed form of the dimer is
modeled into the corresponding position on the open conformation.
(c) Cartoon of a possible model for ATP-switched opening and closing of the N-domain molecular clamp. Binding of ATP to the
N-domains recruits p23, which interacts with the bound ATP and a
site in the C-terminal region of Hsp90, promoting a conformational
change that allows the release of a bound protein or peptide. Hydrolysis of the ATP releases bound p23, and the clamp closes.
Structure of ADP/ATP–Hsp90 N-Domain Complex
73
proven but appears highly likely from the structural data,
although an ADP/ATP mechanism that functions by nucleotide exchange but not hydrolysis cannot be ruled
out. Finally, the conformationaly flexible molecular
clamp previously identified in the structure of the
yeast Hsp90 N-domain would appear to offer an ATPswitchable binding site for accessory proteins or for
client proteins and may also play a role in the intracellular
trafficking of antigenic peptides.
the limits of detection of ITC and consequently have large apparent
errors in measurement.
Graphical Representations
Figures 1a, 1b, 2c, 3, 4, 6a, and 6b were produced using Molscript
(Kraulis, 1991) and Raster3D (Merrit and Murphy, 1994). Figure 1c
was produced using Raster3D and Robert Esnouf’s adaptation of
Molscript (Bobscript), 2a by GRASP (Nicholls et al., 1993), and 2b
by Ligplot (Wallace et al., 1995).
Acknowledgments
Experimental Procedures
Crystal Growth and Data Collection
Tetragonal crystals of Hsp90 N-domain complexes with ATP,
ATPgS, and ADP were grown by vapor diffusion in hanging drops
under very similar conditions to native crystals (Prodromou et al.,
1996) but with the addition of nucleotides (5 mM) and MgCl2 (5 mM)
to the mother liquor. Crystals were stabilized for freezing in a solution
containing 30% glycerol, 90 mM ammonium sulphate, 45 mM sodium succinate (pH 5) and 13.5% polyethylene glycol methyl ester
550. Diffraction data for Hsp90 cocrystals with ATP and ADP were
collected from crystals frozen at 110 K on Station 9.5 (l 5 0.92 Å)
at the SRS, CLRC Daresbury Laboratory, Warrington, UK. The ATP
set consisted of 74,267 reflections collected to 2.0 Å (21,438 unique
reflections, 99.9% complete, Rmerge 5 11.6% with I/sI 5 3.0 in the
final shell). The ADP crystals diffracted further, giving 95,477 reflections to 1.84 Å (27,155 unique, 99.0% complete, Rmerge 5 9.3 with
I/sI 5 3.2 in the final shell). The refined cell of a 5 73.91 Å, c 5
110.95 Å with spacegroup P43 22 was the same for both data sets
and close to that observed for tetragonal crystals of the protein
alone. A third dataset from a cocrystal with ATPgS was collected
locally on a Rigaku/MAR system with CuKa radiation to 2.5 Å (T 5
110 K, 11,273 unique, 99.9% complete, Rmerge 5 6.3 with I/sI 5 2.6
in the final shell).
Structure Refinement
The ATP and ADP data sets were subject to the same refinement
protocol, consisting of X-PLOR (Brünger, 1992) rigid-body refinement of the tetragonal crystal form of the apo yeast Hsp90 N-domain
structure (Prodromou et al., 1997; Brookhaven Protein Databank
accession no. 1AH6) in the new cell, followed by simulated annealing-refinement from 40008C with 258C step cooling. The ATP or ADP
was placed in the clear difference density, and the complex was
subjected to a further 100 cycles of positional refinement. Waters
were added automatically using 2 3 10 cycles of REFMAC (CCP4,
1994) and ARP (Lamzin and Wilson, 1993) (230 for ATP, 360 for
ADP). Final R-factors from REFMAC were: ATP, Rw 5 18.3%, Rfree 5
24.3%; ADP, Rw 5 16.4%, Rfree 5 23.1%. The ATPgS data was refined
using X-PLOR only. All electron density map interpretation and
model building was performed with O (Jones et al., 1991).
Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) gives a complete thermodynamic characterization of an interaction based on the equation
2RT.ln K B 5 DG8 5 DH8 2 TDS8, where R is the gas constant, T is
the absolute temperature, and DG8, DH8, and DS8 are the standard
free-energy, enthalpy, and entropy changes on going from unbound
to bound states, respectively.
The titration experiments were performed using the MSC system
(MicroCal Inc., MA) as described elsewhere (Ladbury and Chowdhry,
1996). All experiments involved injecting 16 aliquots of 15 ml of 1
mM ATP or ADP into 1.3 ml of Hsp90 N-domain at 100 mM at 258C.
All experiments were carried out in 20 mM Tris (pH 7.4) in the presence or absence of 5 mM MgCl 2. The resulting data were fit as
described elsewhere (Wiseman et al., 1989; Ladbury and Chowdhry,
1996) after subtracting the heats of dilution. Heats of dilution were
determined in separate experiments from addition of ATP or ADP
into buffer and buffer into protein. Titration data were fit using a
nonlinear least-squares curve-fitting algorithm with three floating
variables: stoichiometry, binding constant (KB 5 1/KD), and change
of enthalpy of interaction (DH8). The data for ATP binding are close to
We are very grateful to Dale Wigley for making the undeposited
coordinates of DNA gyrase B available to us and to Dietlind Gerloff
and Fred Cohen for drawing our attention to the results of CASP2.
We thank Christine Orengo for assistance with structure alignment,
Roman Laskowski for much assistance with surface analysis and
generation of figures, and Tony Maxwell for very useful discussion.
We are grateful to the Daresbury Laboratory, Warrington, U.K., for
access to the Synchrotron Radiation Source and to the Ludwig
Institute for Cancer Research for provision of X-ray diffraction facilities. J. E. L. is a Wellcome Trust Career Development Fellow. This
work was supported by a Wellcome Trust project grant to P. W. P.
and L. H. P.
Received May 21, 1997; revised June 9, 1997.
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Brookhaven Protein Databank Accession Number
The Brookhaven Protein Databank accession numbers for coordinates of the ADP–Hsp90 and ATP-Hsp90 complexes reported here
are 1AMW and 1AM1, respectively.