Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.     

Amide hydrogen exchange reveals conformational changes in hsp70 chaperones important for allosteric regulation

Hsp70 chaperones assist protein folding processes by a nucleotide-driven cycle of substrate binding and release. Although structural information is available for the isolated nucleotide-binding (NBD) and substrate-binding domains (SBD) in the high affinity conformation, the low affinity conformations and the conformational changes associated with mutual allosteric regulation remained largely enigmatic. By using amide hydrogen exchange in combination with mass spectrometry, we analyzed the Escherichia coli Hsp70 homologue DnaK as full-length protein and its individual domains in the nucleotide-free and ATP-bound conformation. We found a surprising degree of flexibility in both domains. The comparison of the full-length protein with the isolated domains demonstrates a mutual stabilization of both domains. This protection from solvent was most pronounced and in addition was nucleotide-dependent in the lowerbeta-sheet of the SBD and the loop that connects the last beta-strand with helix alphaA. Interestingly, the linker region, which connects NBD and SBD and which is close to the protected loop in the SBD, is solvent-exposed in the absence of nucleotide and completely protected from hydrogen exchange in the presence of ATP. Peptide binding to DnaK.ATP reverts the ATP-induced conformational changes in the linker and selected parts of the NBD. Our data outline a pathway for allosteric interdomain control and suggest an important role of the linker and the base of helix alphaA.     

HSPA1A conformational mutants reveal a conserved structural unit in Hsp70 proteins

BackgroundThe Hsp70 proteins maintain proteome integrity through the capacity of their nucleotide- and substrate-binding domains (NBD and SBD) to allosterically regulate substrate affinity in a nucleotide-dependent manner. Crystallographic studies showed that Hsp70 allostery relies on formation of contacts between ATP-bound NBD and an interdomain linker, accompanied by SBD subdomains docking onto distinct sites of the NBD leading to substrate release. However, the mechanics of ATP-induced SBD subdomains detachment is largely unknown.MethodsHere, we investigated the structural and allosteric properties of human HSPA1A using hydrogen/deuterium exchange mass spectrometry, ATPase assays, surface plasmon resonance and fluorescence polarization-based substrate binding assays.ResultsAnalysis of HSPA1A proteins bearing mutations at the interface of SBD subdomains close to the interdomain linker (amino acids L399, L510, I515, and D529) revealed that this region forms a folding unit stabilizing the structure of both SBD subdomains in the nucleotide-free state. The introduced mutations modulate HSPA1A allostery as they localize to the NBD-SBD interfaces in the ATP-bound protein.ConclusionsThese findings show that residues forming the hydrophobic structural unit stabilizing the SBD structure are relocated during ATP-activated detachment of the SBD subdomains to different NBD-SBD docking interfaces enabling HSPA1A allostery.General significanceMutation-induced perturbations tuned HSPA1A sensitivity to peptide/protein substrates and to Hsp40 in a way that is common for other Hsp70 proteins. Our results provide an insight into structural rearrangements in the SBD of Hsp70 proteins and highlight HSPA1A-specific allostery features, which is a prerequisite for selective targeting in Hsp-related pathologies.     

Allostery in Hsp70 Chaperones Is Transduced by Subdomain Rotations

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's.It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD?Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.     

Crystal structures of the 70-kDa heat shock proteins in domain disjoining conformation

The 70-kDa heat shock proteins (Hsp70s) are highly conserved ATP-dependent molecular chaperones composed of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD) in a bilobate structure. Interdomain communication and nucleotide-dependent structural motions are critical for Hsp70 chaperone functions. Our understanding of these functions remains elusive due to insufficient structural information on intact Hsp70s that represent the different states of the chaperone cycle. We report here the crystal structures of DnaK from Geobacillus kaustophilus HTA426 bound with ADP-Mg(2+)-P(i) at 2.37A and the 70-kDa heat shock cognate protein from Rattus norvegicus bound with ADP-P(i) at 3.5A(.) The NBD and SBD in these structures are significantly separated from each other, and they might depict the ADP-bound conformation. Moreover, a Trp reporter was introduced at the potential interface region between NBD and the interdomain linker of GkDnaK to probe environmental changes. Results from fluorescence measurements support the notion that substrate binding enhances the domain-disjoining behavior of Hsp70 chaperones.     

Interdomain interactions dictate the function of the Candida albicans Hsp110 protein Msi3

Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the “holdase” activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain–domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDβ interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDβ contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the “holdase” activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.     

Structural basis of J cochaperone binding and regulation of Hsp70

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.     

Effect of evolution of the C-terminal region on chaperone activity of Hsp70

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C-terminal α-helical lid subdomain of substrate-binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β-sandwich substrate-binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C-terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C-terminal region from eukaryotic Hsp70 members cannot properly support SBDα–SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα–SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα–SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s.     

The nucleotide‐bound/substrate‐bound conformation of the Mycoplasma genitalium DnaK chaperone

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains—nucleotide binding domain (NBD) and substrate binding domain (SBD)—connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide‐bound/substrate‐bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDβ‐SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.     

Conserved Distal Loop Residues in the Hsp104 and ClpB Middle Domain Contact Nucleotide-binding Domain 2 and Enable Hsp70-dependent Protein Disaggregation

The homologous hexameric AAA⁺ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis.     

Opening of the ADP-bound active site in the ABC transporter ATPase dimer: evidence for a constant contact, alternating sites model for the catalytic cycle

ABC transporters are ubiquitous, ATP-dependent transmembrane pumps. The mechanism by which ATP hydrolysis in the nucleotide-binding domain (NBD) effects conformational changes in the transmembrane domain that lead to allocrite translocation remains largely unknown. A possible aspect of this mechanism was suggested by previous molecular dynamics simulations of the MJ0796 NBD dimer, which revealed a novel, nucleotide-dependent intrasubunit conformational change involving the relative rotation of the helical and catalytic subdomains. Here, we find that in four of five simulations of the ADP/ATP-bound dimer, the relative rotation of the helical and catalytic subdomains in the ADP-bound monomer results in opening of the ADP-bound active site, probably sufficient or close to sufficient to allow nucleotide exchange. We also observe that in all five simulations of the ADP/ATP-bound dimer, the intimate contact of the LSGGQ signature sequence with the ATP gamma-phosphate is weakened by the intrasubunit conformational change within the ADP-bound monomer. We discuss how these results support a constant contact model for the function of the NBD dimer in contrast to switch models, in which the NBDs are proposed to fully disassociate during the catalytic cycle.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

NMR study of nucleotide-induced changes in the nucleotide binding domain of Thermus thermophilus Hsp70 chaperone DnaK: implications for the allosteric mechanism

We present an NMR investigation of the nucleotide-dependent conformational properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein. Conformational changes driven by ATP binding and hydrolysis in the N-terminal NBD are believed to allosterically regulate substrate affinity in the C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs in different nucleotide states have, however, not shown significant structural differences. We have previously reported the NMR assignments of the backbone resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus DnaK in the ADP-bound state. In this study we show, by assigning the NBD with the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the ATP-bound state. Chemical shift difference mapping of the two nucleotide states identified differences in a cluster of residues at the interface between subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP state exhibited a single conformation, whereas the ADP state was in slow conformational exchange between a form similar to the ATP state and another state unique to the ADP-bound form. A model is proposed of the allosteric mechanism based on the nucleotide state altering the balance of a dynamic equilibrium between the open and closed states. The observed chemical shift perturbations were concentrated in an area close to a previously described J-domain binding channel, confirming the importance of that region in the allosteric mechanism.     

Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.     

ATPase Subdomain IA Is a Mediator of Interdomain Allostery in Hsp70 Molecular Chaperones

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other''s activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions.     

Hsp70核苷酸结合域突变体影响酶活的分子动力学模拟研究

分子伴侣蛋白Hsp70氮端核苷酸结合域（NBD, nucleotide-binding domain）的ATP酶活性变化对其行使分子伴侣功能具有重要作用。本文采用分子动力学模拟方法研究酵母分子伴侣Hsp70氮端NBD内残基A17,R23,G32和R167点突变对其ATP酶活性区域构象影响及功能关系。结果表明,突变体A17V,T23H,G32S的ATP结合口袋袋口的loopl（第一个转角,连接p1与p2）结构柔性增强,活性残基T11侧链明显向内移动,从而更加接近ATP的γ-磷酸基团,更容易使ATP水解。这可能蕞终导致ATP酶活性增强,从而引起分子伴侣功能的变化。     

Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70

Hsp70 chaperones are composed of two domains; the 40 kDa N-terminal nucleotide-binding domain (NDB) and the 30 kDa C-terminal substrate-binding domain (SBD). Structures of the SBD from Escherichia coli homologues DnaK and HscA show it can be further divided into an 18 kDa beta-sandwich subdomain, which forms the hydrophobic binding pocket, and a 10 kDa C-terminal three-helix bundle that forms a lid over the binding pocket. Across prokaryotes and eukaryotes, the NBD and beta-sandwich subdomain are well conserved in both sequence and structure. The C-terminal subdomain is, however, more evolutionary variable and the only eukaryotic structure from rat Hsc70 revealed a diverged helix-loop-helix fold. We have solved the crystal structure of the C-terminal 10 kDa subdomain from Caenorhabditis elegans Hsp70 which forms a helical-bundle similar to the prokaryotic homologues. This provides the first confirmation of the structural conservation of this subdomain in eukaryotes. Comparison with the rat structure reveals a domain-swap dimerisation mechanism; however, the C. elegans subdomain exists exclusively as a monomer in solution in agreement with the hypothesis that regions out with the C-terminal subdomain are necessary for Hsp70 self-association.     

A Highlights from MBoC Selection: Role of the loop L4,5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L_4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L_4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L_4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein–bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.     

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23     

Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotide-free Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.