Acta Biochim Biophys Sin 2012, 44: 300– 306 | ª The Author 2012. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gms001.
Advance Access Publication 7 February 2012
Original Article
Structure insights into mechanisms of ATP hydrolysis and the activation of human
heat-shock protein 90
Jian Li1, Lihua Sun1, Chunyan Xu1, Feng Yu1, Huan Zhou1, Yanlong Zhao2, Jian Zhang2, Jianhua Cai3, Cheney Mao3,
Lin Tang1, Yechun Xu4, and Jianhua He1 *
1
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China
Department of Pathophysiology and Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai
Jiao-Tong University, School of Medicine, Shanghai 200025, China
3
Vivabiotech Shanghai Ltd, Shanghai 201203, China
4
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
*Correspondence address. Tel: þ86-21-33933021; Fax: þ86-21-33933021; E-mail: [email protected]
2
The activation of molecular chaperone heat-shock protein
90 (Hsp90) is dependent on ATP binding and hydrolysis,
which occurs in the N-terminal domains of protein. Here,
we have determined three crystal structures of the N-terminal domain of human Hsp90 in native and in complex
with ATP and ATP analog, providing a clear view of the
catalytic mechanism of ATP hydrolysis by Hsp90.
Additionally, the binding of ATP leads the N-terminal
domains to be an intermediate state that could be used to
partially explain why the isolated N-terminal domain of
Hsp90 has very weak ATP hydrolytic activity.
Keywords
human Hsp90; N-terminal domain; ATP
hydrolysis; catalytic mechanism
Received: October 28, 2011
Accepted: November 30, 2011
Introduction
The molecular chaperone heat-shock protein 90 (Hsp90),
which is upregulated in response to stress, is essential for
folding, maturation, stabilization, and localization events of
the chaperone’s client proteins that are involved in cell
cycle regulation, signal transduction, and cell growth regulation. Hsp90 can be divided into three domains. The
highly conserved N-terminal domain contains an unusually
shaped ATP-binding cleft and is thus responsible for the
catalytic activity of Hsp90. The middle domain containing
a large hydrophobic surface helps the folding of its client
proteins, while the C-terminal domain is critical for the formation of Hsp90 homodimer [1–3]. The function of Hsp90
is tightly associated with the binding and hydrolysis of
ATP as well as the dimerization of the chaperone [4,5].
ATP binding in the N-terminal domain of Hsp90 drives a
structural rearrangement and changes the surface properties
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 300
of the N-terminal domain, which induces the dimerization
of the N-terminal domains and influences the affinity of
Hsp90 for client proteins [6,7]. ATP-mediated closing of
the interprotomer space drives client proteins remodeling.
Once ATP hydrolysis occurs, a conversion of Hsp90 to its
ADP bound state leads to the release of client proteins. In
addition, the release efficiency of client proteins might be
enhanced by the co-chaperones p23 [8]. Biochemical investigations of the ATP/ADP cycle revealed that the catalytic
efficiency of Hsp90 is tightly regulated by the dimerization
of the N-terminal as well as the association of the
N-terminal domain with the middle domain [9].
The first X-ray structure of isolated N-terminus of
human Hsp90 (Hsp90N) was determined by Stebbins et al.
[10] and the complex structure of Hsp90N –ADP was
reported by Obermann et al. [11]. In 2006, a crystal structure of the full-length yeast Hsp90-AMPPNP was resolved
[12]. Shiau et al. [7] described the cryo-electron microscopy as well as X-ray structures of nucleotide-free,
AMPPNP-bound, and ADP-bound states of HtpG
(Escherichia coli Hsp90 ortholog), which were similar in
both size and detailed shape to the yeast Hsp90-AMPPNP
structure. In addition, a series of X-ray crystal structures of
human Hsp90N inhibitors have been reported [13–15].
Although the structures of the isolated N-terminus of
human Hsp90 and the protein in complex with ATPgS
have been reported [11,16], it is still not clear as to how
Hsp90 catalytically hydrolyzes ATP to produce ADP at the
atomic level. To understand the structural and mechanistic
consequences of nucleotide binding to human Hsp90, we
have determined three crystal structures of the N-terminal
domain of human Hsp90 in apo and in complex with ATP
and adenosine 50 -(b,g-methylene) triphosphate (AMPPCP).
It is the first time to capture the real human Hsp90N –ATP
complex structure and describe the binding of g-phosphate
Mechanisms of ATP hydrolysis of human heat-shock protein 90
of ATP and the water molecule that mediates the ATP hydrolysis to Hsp90N. In addition, the changes in the interactions between R46-E47 and R46-S129 during ATP binding
were analyzed in detail, which explain why the isolated
N-domain of Hsp90 has very weak ATP hydrolytic activity. The structural data presented here thus provided new
insights into the complicated and subtle mechanism of
ATP hydrolysis by Hsp90 at atomic level.
Materials and Methods
Protein purification and crystallization
A cDNA fragment encoding the N-terminal domain of
human Hsp90 (residues 9–236), Hsp90N, was cloned into
a pET28 vector. The recombinant plasmid pET28-Hsp90N
was transferred into E. coli strain BL21 (DE3) for overexpression (Invitrogen, Carlsbad, USA). Bacterial cultures
were grown in LB medium at 378C to an OD600 of 0.6–
0.8, and 0.2 mM isopropyl-b-D-thiogalactopyranoside was
then added for further growth at 308C for another 5 h. The
harvested cells were resuspended in lysis buffer (20 mM
Tris/300 mM NaCl/5 mM b-mercaptoethanol/10% glycerol, pH 7.5) and sonicated. The resulted suspension was
centrifuged and the supernatant was passed through a
nickel-bead column (GE Healthcare, Piscataway, USA).
The column was then washed with 20 mM imidazole/
300 mM NaCl/20 mM Tris/5 mM b-mercaptoethanol/10%
glycerol, pH 7.5 (wash buffer), until no further protein was
eluted. The recombinant Hsp90N was then eluted with
100 mM imidazole/300 mM NaCl/20 mM Tris/5 mM
b-mercaptoethanol/10% glycerol, pH 7.5 (elution buffer).
The eluted proteins were concentrated (Millipore, Billerica,
USA) and then injected into a 120 ml Hiload Superdex75
column (GE Healthcare) from which Hsp90N was eluted
with 20 mM Tris/300 mM NaCl/5 mM b-mercaptoethanol/
10% glycerol, pH 7.5. The fractions containing Hsp90N
were collected and concentrated to 20 mg/ml for crystallization. The purity of protein was assessed by SDS-PAGE
to be .95%.
Crystallization of apo Hsp90N was performed at 48C
using the hanging drop vapor-diffusion method, by mixing
equal volumes of the protein and of the precipitant [20%–
25% (w/v) PEG 2000 monomethyl ether/200 mM magnesium chloride/100 mM sodium cacodylate, pH 6.5]. The
complexes of Hsp90N with two nucleotides were prepared
by mixing the protein solution (20 mg/ml) with a five-fold
molar excess of nucleotides. Co-crystallization of Hsp90N
with nucleotides was carried out at 48C using the hanging
drop vapor-diffusion method, by mixing the solution of the
complexes with an equal volume of a precipitant solution.
The precipitant solutions for Hsp90N –AMPPCP and
Hsp90N –ATP complexes are 20%–25% PEG4000/
200 mM magnesium chloride/100 mM Tris at pH 8.5 and
15%–20% PEG3350/5 mM magnesium chloride/500 mM
ammonium phosphate/100 mM Bis-Tris at pH 6.5,
respectively.
Data collection, structure determination, and
refinement
After 3–4 days, crystals were grown and then mounted and
flash-frozen in liquid nitrogen for diffraction test and data
collection. All data sets were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation
Facility (SSRF, Shanghai, China) and were processed with
the HKL2000 software package [17]. The structures were
solved by molecular replacement, using the PHENIX [18].
The search model used for the crystals of apo and the
complex was the previously reported structure of the
Hsp90-GBD (PDB code 1YES), the structures were refined
using PHENIX [18]. With the aid of the program Coot
[19], nucleotides, water molecules, and others were fitted
into to the initial Fo –Fc map. Figures of interactions
between Hsp90N and nucleotides were generated using
LIGPLOT [20]. The complete statistics, as well as the
quality of the solved structures, are shown in Table 1.
Results
Crystal structures of Hsp90N – nucleotides complexes
Crystal structures of Hsp90N in the absence and presence
of ATP and ATP analog (Fig. S1) with different crystal
packing patterns have been solved. We crystallized the
native Hsp90N, and collected a data set with a higher resolution (1.2 Å) than those previously deposited in PDB, so
as to exclude the influences of different protein sequences,
purification protocols, crystallization conditions, and structure refinement procedures on the crystal structure
obtained. The highest resolution for apo Hsp90N, Hsp90N –
AMPPCP, and Hsp90N –ATP structures are 1.20, 1.24, and
2.20 Å, respectively (PDB code 3T0H, 3T10, and 3T0Z,
respectively). The difference Fourier maps shown in
Fig. 1 clearly demonstrated, that ligands were fully bound
into Hsp90N in the two complex structures.
Interactions between Hsp90N and nucleotides
To our knowledge, it is the first time to solve the real
human ATP –Hsp90N complex structure. To validate the
binding of ATP with Hsp90N, we also determined the
structures of Hsp90N in complex with AMPPCP. Although
the two independently refined structures were at different
resolution levels and with different crystal packing patterns,
there is no significant conformational change observed
between the two protein structures [Fig. 2(A)]. The nucleotides are also overlapped well [Fig. 2(B)]. Additionally,
superimposition of Hsp90N –ATP with Hsp90N –ADP
(PDB code 1BYQ) structures revealed that the ATP
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 301
Mechanisms of ATP hydrolysis of human heat-shock protein 90
Table 1 Statistics for data processing and model refinement of human Hsp90N – nucleotide complexes
Space group
I222
P21
P21
a, b, c (Å)
a, b, g (8)
Resolution (Å)
Rsym (Rmerge)
I/s (I)
Completeness (%)
Redundancy
Resolution (Å)
Reflections in working set
Reflections in test set
Rwork (Rfree)
Protein
Ligand/ion
Water
Mean temperature factor (Å2)
Bond lengths (Å)
Bond angles (8)
65.29, 88.93, 99.67
90.00, 90.00, 90.00
1.20 (1.22– 1.20)
0.065 (0.640)
929.5/20.9
99.0 (91.6)
13.3 (6.6)
34.483– 1.20
89825
3825
0.1421 (0.1518)
3290
53.17, 44.97, 53.85
90.00, 115.15, 90.00
1.24 (1.26 – 1.24)
0.054 (0.509)
892.2/20.8
97.6 (97.2)
7.2 (6.3)
26.819– 1.24
63893
2526
0.1576 (0.1720)
3374
ACP/MG
242
19.2
0.012
1.5
50.71, 46.78, 53.22
90.00, 111.59, 90.00
2.20 (2.24– 2.20)
0.165 (0.528)
35.9/1.7
99.0 (100.0)
7.1 (6.4)
49.488 – 2.20
11954
660
0.1968 (0.2559)
1680
ATP/MG/GOL
78
21.0
0.0081
1.217
140
18.5
0.0098
1.355
Values in parentheses are for the highest-resolution shell.
Figure 1 Electron density maps (2Fo – Fc) of the two bound
nucleotides The maps were contoured at a level of 1.0 s; carbon,
nitrogen, oxygen, and phosphorus atoms of residues are shown in green,
blue, red, and magenta, respectively.
occupied the same location as ADP except the additional
g-phosphate group of ATP formed more interactions with
Hsp90N (Fig. 3).
Since the crystal of Hsp90N –AMPPCP complex diffracted to 1.24 Å, which has the higher resolution than the
Hsp90N –ATP structures, the interactions between
AMPPCP and Hsp90N were analyzed in more detail
(Fig. 4). As shown in Fig. 4(C), the N6 atom of the
adenine group not only forms a direct hydrogen bond (HB)
with the carboxyl group of D93, which is most critical for
recognition of Hsp90 to the nucleotide binding, but also
interacts with L48 and S52 through water-mediated HBs.
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 302
Figure 2 Comparison of the crystal structures of Hsp90N – ATP with
Hsp90N – AMPPCP complexes (A) Ribbon superimposition of the two
crystal structures of Hsp90N – AMPPCP and Hsp90N –ATP complexes
shown with different colors. (B) The two nucleotides extracted from
complexes shown in (A) in a same view.
Mechanisms of ATP hydrolysis of human heat-shock protein 90
Figure 3 Comparison of the crystal structures of Hsp90N – ATP with Hsp90N – ADP complexes. (A) Superimposition of Hsp90N – ATP (green) and
Hsp90N – ADP complex (PDB code 1BYQ, magenta). (B) Enlarged view of the superimposed ATP and ADP as well as their surroundings.
Figure 4 Interactions between Hsp90N and AMPPCP (A) Surface representation of AMPPCP bound in the pocket of Hsp90N. (B) Interactions of
magnesium ion, nucleotide with residues of Hsp90N. The hydrogen and coordination bonds are shown by black and cyan dashed lines, respectively.
(C) Schematic diagrams of the interactions between Hsp90N and AMPPCP. HBs are shown as green dashed lines. Spiked residues form hydrophobic
interactions with AMPPCP. Water molecules are shown as cyan spheres. This picture was made with the program LIGPLOT [20].
The N7 atom of the adenine ring interacts with residue
N51 via water-mediated HBs while the N1 atom of the
adenine ring forms water-mediated HBs with residues D93,
G97, and T184. The interactions formed between Hsp90N
and the ribose ring of AMPPCP include a direct HB
between atom O2 and residue N106, and two watermediated HBs of atom O1 with positive charged side chain
of K58 and K112. The a-phosphate group forms direct
HBs with N51 and F138, and water-mediated HBs with
E47, N51, and G135. The b-phosphate group forms watermediated HBs with N51 and D54. The g-phosphate group
is surrounded by a loop at the C-terminal hinge of the ATP
lid that is mainly constituted by residues 111–134, and
forms direct HBs with the backbone of G137 and watermediated HBs with side chain of E47, N51, and D54. A
magnesium ion is octahedrally coordinated with oxygen
atoms of the a-, b-, and g-phosphate groups, the side
chain of N51, and two water molecules [Fig. 4(B)]. As for
the Hsp90N –ATP structures, the interactions between
Hsp90N and ATP are very similar to those in the Hsp90N –
AMPPCP (Fig. S2). The interactions of the magnesium ion
with nucleotide and residues of Hsp90N are also similar to
those observed in Hsp90N –AMPPCP complex.
Constitution of the catalytic center of the human Hsp90
ATP hydrolysis
Examination of the residues surrounding the g-phosphate
of the bound ATP in Hsp90N revealed that E47 is ideally
located to promote nucleophilic attack by the key water
molecule to the g-phosphate of ATP. The water molecule
forms HBs not only with the carboxyl group of E47 but
also with the two oxygen atoms of the g-phosphate group.
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Mechanisms of ATP hydrolysis of human heat-shock protein 90
Figure 5 Details of the catalytic center of the human Hsp90 with
ATP The coordinates of all except for R400 are extracted from
Hsp90N – ATP structure. The coordinates of R400 are those of R380 in
the structure of 2CG9 after superimposition to the Hsp90N – ATP structure
by fitting the N-terminal domain only.
Because of the strong HB interaction formed between R46
and E47, R46 is also considered to be involved in the catalytic process (Fig. 5) [21,22]. In addition, it was reported
that R400 is also essential for Hsp90 to perform the
ATPase activity and function [22]. Since the whole structure of human Hsp90 is still not available, the full-length
structure of yeast Hsp90 was taken as a model due to the
high sequence similarity and the almost identical residues
involved in nucleotide hydrolysis between the two molecules (Fig. S3). In fact, the ATP observed in our human
Hsp90N –ATP complex structure occupies the same position as AMPPNP bound in the full length of yeast Hsp90
(Fig. 6) [12]. Therefore, the coordinates of residue R400
(corresponding to R380 in yeast) as well as the huge conformational change of the whole chaperone associated with
ATP/ADP cycle discussed below were based on the fulllength structure of yeast Hsp90 but substituted with human
Hsp90 sequence. Besides the key residues, a magnesium
ion is always octahedrally coordinated with the nucleotide
as well as the protein in two complexes. The coordination
bonds between magnesium ion and oxygen atoms are
varying from 2.0 to 2.2 Å (Fig. 5).
Analysis of crystal structures of Hsp90N and Hsp90N –
ATP complexes
The first complex structure of Hsp90N –ATP provides the
structural basis to further investigate the activated conformational change of Hsp90 after ATP binding.
Superimposition of Hsp90N –ATP complex with the native
Hsp90N structures shows that the H1, H4, H5, and L6 of
Hsp90N undergo a conformational rearrangement in response to the bound ATP (Fig. 7). In particular, H4 reorients and moves towards H1, and L6 flaps 1808 from
inward to outward the ATP-binding site.
Detailed analysis of the interactions shows that, without
ATP binding, the strong HB interactions are formed
between residues E25 and K112, and between residues
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 304
Figure 6 Comparison of the crystal structures of human Hsp90N –
ATP with yeast Hsp90 –AMPPNP complexes (A) Superimposition of
human Hsp90N – ATP and yeast Hsp90– AMPPNP (PDB code 2CG9)
complexes. The g-phosphate of ATP and AMPPNP is orientated in the
same direction. (B) Enlarged view of the superimposed ATP and
AMPPNP as well as their surroundings. The human Hsp90N is shown in
magenta, the yeast Hsp90 is shown in green, and the catalytic loop
encoding residue R380 is shown in blue.
Q23 and N106 as well [Fig. 8(A)]. Those interactions
reduce the flexibility of L6 and hinder the flapping of the
ATP lid in the apo structure. However, in the Hsp90N –
ATP complex, these two HBs are broken and new interactions are formed. Residue N106 forms one new HB with
atom O1 of ATP directly and K112 forms two new HBs
with atom O2 and b-phosphate oxygen of ATP through
two water molecules [Fig. 8(B)]. Therefore, the binding of
ATP induces the ATP lid to be a more open conformation
but with less interactions formed between the ATP lid and
the rest part of the protein, which may play an important
role in regulating the ATP/ADP cycle.
Discussion
Catalytic mechanism of ATP hydrolysis by Hsp90
The g-phosphate group of ATP, R46, E47, R400, Mg2þ,
and a water molecule constitute the catalytic center for
ATP hydrolysis (Fig. 5). The g-phosphate oxygen atoms
form coordination bonds with Mg2þ, direct HBs with the
backbone of residues G132 and G137, water-mediated HBs
with the polar groups of residues E47, and strong HBs (salt
Mechanisms of ATP hydrolysis of human heat-shock protein 90
Figure 7 Comparison of the crystal structures of Hsp90N with Hsp90N – ATP complexes Superimposition of the structures of apo Hsp90N ( pink)
and Hsp90N – ATP (green).
Figure 8 Details of the residue interactions of Hsp90N and Hsp90N –ATP complexes (A) In the apo structure of Hsp90N, two HB interactions
formed between the ATP lid and helix H1: K112-E25 and N106-Q23. (B) In Hsp90N – ATP complex, residues K112 and N106 form HB directly or
indirectly with the bound ATP. (C) In the apo structure of Hsp90N, there is no HB in D127-N40 and S129-R46 residue pairs. (D) In Hsp90N –ATP
complex, residues E47 and S129 form direct HBs with R46, and N40 forms a direct HB with D127.
bridge) with positive charged side chain of R400.
Accordingly, a detailed catalytic mechanism is proposed to
demonstrate the hydrolysis of ATP catalyzed by Hsp90
(Fig. 5). Firstly, R46 forms strong HB (salt bridge) with
the carboxyl group of E47, which polarizes E47 and makes
it as a general base. Then, the polarized E47 nucleophilic
attacks the water molecule and absorbs the proton from the
water molecule, providing the electron to the water molecule and make it more negatively charged. Finally, the
negative-charged water molecule nucleophilic attacks the
phosphorus atom of the g-phosphate, the covalent bond
between b- and g-phosphate is broken, and the products, a
hydrogen phosphate and ADP, are formed. Magnesium ion
is universally required for ATP hydrolysis by Hsp90
[23–25], and the role of the magnesium ion is suggested to
stabilize the intermediate state of catalytic reaction. After
hydrolysis reaction finishes, the interaction between R400
and the nucleotide gets broken. The breaking the interaction between R400 and the nucleotide, and the subsequent release of g-phosphate after hydrolysis may induce
the dissociation of the N-terminal and the middle domains
in each monomer as well as the dissociation of two
N-terminal domains, allowing the release of the client
protein [6,7].
Weak ATP hydrolytic activity of Hsp90N
The electrostatic interactions of Q23-N106 and E25-K112
residue pairs were interrupted due to the ATP binding, releasing the restriction on the conformational change of the
H1 [Fig. 8(A,B)]. The increased flexibility of H1 makes it
in high possibility to swing and form interactions with the
N-terminal domain of the other monomer which was
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 305
Mechanisms of ATP hydrolysis of human heat-shock protein 90
shown in the dimer of whole yeast Hsp90 structure [24,26].
It is thus suggested that conformation of the Hsp90N –ATP
complex is an intermediate state for the whole chaperone
activation.
The ATP binding drives the preliminary formation of the
catalytic center for ATP hydrolysis. Residue R46 firstly
moves close to E47, as the length of the HB between these
two residues shortens from 3.1 Å in the apo structure to
2.8 Å in Hsp90N –ATP complex. Meanwhile, the
g-phosphate of the bound nucleotide forms HBs with E47
through water molecules and the positive-charged residue
R46 forms HB not only with E47 but also with S129
[Fig. 8(C,D)]. The HB formed between S129 and R46 partially absorbs the positive charge of R46, weakening the
polarization of E47 by R46. This, in addition to the missed
interaction of R400 with the g-phosphate of ATP, could
explain why the isolated N-domain of Hsp90 has very
weak ATP hydrolytic activity.
Supplementary Data
Supplementary data are available at ABBS online.
Funding
This work was supported by the grants from the National
Program on Key Basic Research Project (2011CB911102),
Shanghai Institute of Applied Physics, Chinese
Academy of Sciences (O95501C061) and the Important
National Science & Technology Specific Projects
(2010ZX09401-401).
References
1 Johnson JL, Halas A and Flom G. Nucleotide-dependent interaction of
Saccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1,
Cpr6, and Sba1. Mol Cell Biol 2007, 27: 768 – 776.
2 Dollins DE, Immormino RM and Gewirth DT. Structure of unliganded
GRP94, the endoplasmic reticulum Hsp90. J Biol Chem 2005, 280:
30438– 30447.
3 Wright L, Barril X, Dymock B, Sheridan L, Surgenor A, Beswick M and
Drysdale M, et al. Structure– activity relationships in purine-based inhibitor binding to HSP90 isoforms. Chem Biol 2004, 11: 775– 785.
4 Ratzke C, Mickler M, Hellenkamp B, Buchner J and Hugel T. Dynamics
of heat shock protein 90 C-terminal dimerization is an important part of its
conformational cycle. Proc Natl Acad Sci USA 2010, 107: 16101– 16106.
5 Immormino RM, Dollins DE, Shaffer PL, Soldano KL, Walker MA and
Gewirth DT. Ligand-induced conformational shift in the N-terminal
domain of GRP94, an Hsp90 chaperone. J Biol Chem 2004, 279:
46162– 46171.
Acta Biochim Biophys Sin (2012) | Volume 44 | Issue 4 | Page 306
6 McLaughlin SH, Ventouras LA, Lobbezoo B and Jackson SE.
Independent ATPase activity of Hsp90 subunits creates a flexible assembly
platform. J Mol Biol 2004, 344: 813 –826.
7 Shiau AK, Harris SF, Southworth DR and Agard DA. Structural analysis
of E. coli hsp90 reveals dramatic nucleotide-dependent conformational
rearrangements. Cell 2006, 127: 329– 340.
8 Young JC and Hartl FU. Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23. EMBO J 2000, 19:
5930– 5940.
9 Richter K, Reinstein J and Buchner J. N-terminal residues regulate the
catalytic efficiency of the Hsp90 ATPase cycle. J Biol Chem 2002, 277:
44905– 44910.
10 Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU and Pavletich
NP. Crystal structure of an Hsp90-geldanamycin complex: targeting of a
protein chaperone by an antitumor agent. Cell 1997, 89: 239– 250.
11 Obermann WM, Sondermann H, Russo AA, Pavletich NP and Hartl FU.
In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis. J Cell Biol 1998, 143: 901– 910.
12 Ali MM, Roe SM, Vaughan CK, Meyer P, Panaretou B, Piper PW and
Prodromou C, et al. Crystal structure of an Hsp90-nucleotide-p23/Sba1
closed chaperone complex. Nature 2006, 440: 1013 –1017.
13 Jez JM, Chen JC, Rastelli G, Stroud RM and Santi DV. Crystal structure
and molecular modeling of 17-DMAG in complex with human Hsp90.
Chem Biol 2003, 10: 361 – 368.
14 Dymock BW, Barril X, Brough PA, Cansfield JE, Massey A, McDonald E
and Hubbard RE, et al. Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design. J Med
Chem 2005, 48: 4212– 4215.
15 Vallée F, Carrez C, Pilorge F, Dupuy A, Parent A, Bertin L and
Thompson F, et al. Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic imidazo[4,5-c]pyridines as potent inhibitors of the Hsp90 molecular chaperone. J Med Chem 2011, 54:
7206– 7219.
16 Prodromou C, Roe SM, O’Brien R, Ladbury JE, Piper PW and Pearl LH.
Identification and structural characterization of the ATP/ADP-binding site
in the Hsp90 molecular chaperone. Cell 1997, 90: 65– 75.
17 Otwinowski Z and Minor W. Processing of X-ray diffraction data collected
in oscillation mode. Methods Enzymol 1997, 276: 307 – 326.
18 Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N and
Headd JJ, et al. PHENIX: a comprehensive Python-based system for
macromolecular structure solution. Acta Crystallogr D 2010, 66: 213 – 221.
19 Emsley P and Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D 2004, 60: 2126– 2132.
20 Wallace AC, Laskowski RA and Thornton JM. LIGPLOT: a program to
generate schematic diagrams of protein– ligand interactions. Protein Eng
1995, 8: 127 – 134.
21 Jackson AP and Maxwell A. Identifying the catalytic residue of the
ATPase reaction of DNA gyrase. Proc Natl Acad Sci USA 1993, 90:
11232– 11236.
22 Chen B, Zhong D and Monteiro A. Comparative genomics and evolution
of the HSP90 family of genes across all kingdoms of organisms. BMC
Genom 2006, 7: 156.
23 Tomaszek T and Schuster SM. Hydrolysis of adenyl-5-yl imidodiphosphate
by beef heart mitochondrial ATPase. J Biol Chem 1986, 261: 2264– 2269.
24 Corbett KD and Berger JM. Structure of the ATP-binding domain of
Plasmodium falciparum Hsp90. Proteins 2010, 78: 2738– 2744.
25 Wigley DB, Davies GJ, Dodson EJ, Maxwell A and Dodson G. Crystal
structure of an N-terminal fragment of the DNA gyrase B protein. Nature
1991, 351: 624 –629.
26 Richter K, Moser S, Hagn F, Friedrich R, Hainzl O, Heller M and Schlee
S, et al. Intrinsic inhibition of the Hsp90 ATPase activity. J Biol Chem
2006, 281: 11301– 11311.