Differences in conformational dynamics within the Hsp90 chaperone family reveal mechanistic insights.
ABSTRACT: The molecular chaperones of the Hsp90 family are essential in all eukaryotic cells. They assist late folding steps and maturation of many different proteins, called clients, that are not related in sequence or structure. Hsp90 interaction with its clients appears to be coupled to a series of conformational changes. Using hydrogen exchange mass spectrometry (HX-MS) we investigated the structural dynamics of human Hsp90? (hHsp90) and yeast Hsp82 (yHsp82). We found that eukaryotic Hsp90s are much more flexible than the previously studied Escherichia coli homolog (EcHtpG) and that nucleotides induce much smaller changes. More stable conformations in yHsp82 are obtained in presence of co-chaperones. The tetratricopeptide repeat (TPR) domain protein Cpr6 causes a different amide proton protection pattern in yHsp82 than the previously studied TPR-domain protein Sti1. In the simultaneous presence of Sti1 and Cpr6, protection levels are observed that are intermediate between the Sti1 and the Cpr6 induced changes. Surprisingly, no bimodal distributions of the isotope peaks are detected, suggesting that both co-chaperones affect both protomers of the Hsp90 dimer in a similar way. The cochaperones Sba1 was found previously in the crystal structure bound to the ATP hydrolysis-competent conformation of Hsp90, which did not allow to distinguish the mode of Sba1-mediated inhibition of Hsp90's ATPase activity by stabilizing the pre- or post-hydrolysis step. Our HX-MS experiments now show that Sba1 binding leads to a protection of the ATP binding lid, suggesting that it inhibits Hsp90's ATPase activity by slowing down product release. This hypothesis was verified by a single-turnover ATPase assay. Together, our data suggest that there are much smaller energy barriers between conformational states in eukaryotic Hsp90s than in EcHtpG and that co-chaperones are necessary in addition to nucleotides to stabilize defined conformational states.
Project description:Hsp90 is a highly conserved molecular chaperone important for the activity of many client proteins. Hsp90 has an N-terminal ATPase domain (N), a middle domain (M) that interacts with clients and a C-terminal dimerization domain (C). "Closing" of dimers around clients is regulated by ATP binding, co-chaperones, and post-translational modifications. ATP hydrolysis coincides with release of mature client and resetting the reaction cycle. Humans have two Hsp90s: hHsp90? and hHsp90?. Although 85% identical, hHsp90? supports Hsp90 function in yeast much better than hHsp90?. Determining the basis of this difference would provide important insight into functional specificity of seemingly redundant Hsp90s, and the evolution of eukaryotic Hsp90 systems and clientele. Here, we found host co-chaperones Sba1, Cpr6 and Cpr7 inhibited hHsp90? function in yeast, and we identified mutations clustering in the N domain that considerably improved hHsp90? function in yeast. The strongest of these rescuer mutations accelerated nucleotide-dependent lid closing, N-M domain docking, and ATPase. It also disrupted binding to Sba1, which prolongs the closed state, and promoted N-M undocking and lid opening. Our data suggest the rescuer mutations improve function of hHsp90? in yeast by accelerating return to the open state. Our findings imply hHsp90? occupies the closed state too long to function effectively in yeast, and define an evolutionarily conserved region of the N domain involved in resetting the Hsp90 reaction cycle.
Project description:Heat-shock protein 90 (Hsp90) of Saccharomyces cerevisiae is an abundant essential eukaryotic molecular chaperone involved in the activation and stabilization of client proteins, including several transcription factors and oncogenic kinases. Hsp90 undergoes a complex series of conformational changes and interacts with partner co-chaperones such as Sba1, Cpr6, Cpr7, and Cns1 as it binds and hydrolyzes ATP. In the absence of nucleotide, Hsp90 is dimerized only at the carboxy-terminus. In the presence of ATP, Hsp90 also dimerizes at the amino-terminus, creating a binding site for Sba1. Truncation of a charged linker region of yeast Hsp90 (Hsp82?linker) was known to disrupt the ability of Hsp82 to undergo amino-terminal dimerization and bind Sba1. We found that yeast expressing Hsp82?linker constructs exhibited a specific synthetic lethal phenotype in cells lacking CPR7. The isolated tetratricopeptide repeat domain of Cpr7 was both necessary and sufficient for growth in those strains. Cpr6 and Cpr7 stably bound the carboxy-terminus of wild-type Hsp82 only in the presence of nonhydrolyzable ATP and formed an Hsp82-Cpr6-Cpr7 ternary complex. However, in cells expressing Hsp82?linker or lacking CPR7, Cpr6 was able to bind Hsp82 in the presence or absence of nucleotide. Overexpression of CNS1, but not of other co-chaperones, in cpr7 cells restored nucleotide-dependent Hsp82-Cpr6 interaction. Together, our results suggest that the in vivo functions of Cpr7 include modulating Hsp90 conformational changes, mediating proper signaling of the nucleotide-bound state to the carboxy-terminus of Hsp82, or regulating Hsp82-Cpr6 interaction.
Project description:In eukaryotic cells, Hsp90 chaperones assist late folding steps of many regulatory protein clients by a complex ATPase cycle. Binding of clients to Hsp90 requires prior interaction with Hsp70 and a transfer reaction that is mediated by the co-chaperone Sti1/Hop. Sti1 furthers client transfer by inhibiting Hsp90's ATPase activity. To better understand how Sti1 prepares Hsp90 for client acceptance, we characterized the interacting domains and analysed how Hsp90 and Sti1 mutually influence their conformational dynamics using hydrogen exchange mass spectrometry. Sti1 stabilizes several regions in all three domains of Hsp90 and slows down dissociation of the Hsp90 dimer. Our data suggest that Sti1 inhibits Hsp90's ATPase activity by preventing N-terminal dimerization and docking of the N-terminal domain with the middle domain. Using crosslinking and mass spectrometry we identified Sti1 segments, which are in close vicinity of the N-terminal domain of Hsp90. We found that the length of the linker between C-terminal dimerization domain and the C-terminal MEEVD motif is important for Sti1 association rates and propose a kinetic model for Sti1 binding to Hsp90.
Project description:The abundant molecular chaperone Hsp90 is essential for the folding and stabilization of hundreds of distinct client proteins. Hsp90 is assisted by multiple cochaperones that modulate Hsp90's ATPase activity and/or promote client interaction, but the in vivo functions of many of these cochaperones are largely unknown. We found that Cpr6, Cpr7, and Cns1 interact with the intact ribosome and that Saccharomyces cerevisiae lacking CPR7 or containing mutations in CNS1 exhibited sensitivity to the translation inhibitor hygromycin. Cpr6 contains a peptidyl-prolyl isomerase (PPIase) domain and a tetratricopeptide repeat (TPR) domain flanked by charged regions. Truncation or alteration of basic residues near the carboxy terminus of Cpr6 disrupted ribosome interaction. Cns1 contains an amino-terminal TPR domain and a poorly characterized carboxy-terminal domain. The isolated carboxy-terminal domain was able to interact with the ribosome. Although loss of CPR6 does not cause noticeable growth defects, overexpression of CPR6 results in enhanced growth defects in cells expressing the temperature-sensitive cns1-G90D mutation (the G-to-D change at position 90 encoded by cns1). Cpr6 mutants that exhibit reduced ribosome interaction failed to cause growth defects, indicating that ribosome interaction is required for in vivo functions of Cpr6. Together, these results represent a novel link between the Hsp90 molecular-chaperone machine and protein synthesis.
Project description:Heat Shock Protein 90 (Hsp90) is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes) to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast), Aha1, and P23 (Sba1 in yeast) were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that the toxic sterols eburicol and 14?-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.
Project description:The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR) domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a 'carboxylate clamp'. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC)-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1), DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays. These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.
Project description:Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90s conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.
Project description:The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.
Project description:Hsp90 is critical for the regulation and activation of numerous client proteins critical for diverse functions such as cell growth, differentiation, and reproduction. Cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 function or direct Hsp90 to interact with specific client proteins. Little is known about how Hsp90 complexes vary between different organisms and how this affects the scope of clients that are activated by Hsp90. This study determined whether ten distinct Hsp90 co-chaperones were encoded by genes in 19 disparate eukaryotic organisms. Surprisingly, none of the co-chaperones were present in all organisms. The co-chaperone Hop/Sti1 was most widely dispersed (18 out of 19 species), while orthologs of Cdc37, which is critical for the stability and activation of diverse protein kinases in yeast and mammals, were identified in only nine out of 19 species examined. The organism with the smallest proteome, Encephalitozoon cuniculi, contained only three of these co-chaperones, suggesting a correlation between client diversity and the complexity of the Hsp90 co-chaperone machine. Our results suggest co-chaperones are critical for cytosolic Hsp90 function in vivo, but that the composition of Hsp90 complexes varies depending on the specialized protein folding requirements of divergent species.
Project description:In eukaryotes, the molecular chaperones Hsp90 and Hsp70 are connected via the co-chaperone Sti1/Hop, which allows transfer of clients. Here, we show that the basic functions of yeast Sti1 and human Hop are conserved. These include the simultaneous binding of Hsp90 and Hsp70, the inhibition of the ATPase activity of Hsp90, and the ability to support client activation in vivo. Importantly, we reveal that both Hop and Sti1 are subject to inhibitory phosphorylation, although the sites modified and the influence of regulatory phosphorylation is species specific. Phospho-mimetic variants have a reduced ability to activate clients in vivo and different affinity for Hsp70. Hop is more tightly regulated, as phosphorylation affects also the interaction with Hsp90 and induces structural rearrangements in the core part of the protein.