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ABSTRACT: 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.
The EMBO Journal 02/2012; 31(6):1518-28. · 9.20 Impact Factor
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ABSTRACT: Hsp90 is an essential and highly conserved modular molecular chaperone whose N and middle domains are separated by a disordered region termed the charged linker. Although its importance has been previously disregarded, because a minimal linker length is sufficient for Hsp90 activity, the evolutionary persistence of extensive charged linkers of divergent sequence in Hsp90 proteins of most eukaryotes remains unexplained. To examine this question further, we introduced human and plasmodium native and length-matched artificial linkers into yeast Hsp90. After evaluating ATPase activity and biophysical characteristics in vitro, and chaperone function in vivo, we conclude that linker sequence affects Hsp90 function, cochaperone interaction, and conformation. We propose that the charged linker, in addition to providing the flexibility necessary for Hsp90 domain rearrangements--likely its original purpose--has evolved in eukaryotes to serve as a rheostat for the Hsp90 chaperone machine.
Proceedings of the National Academy of Sciences 02/2012; 109(8):2937-42. · 9.68 Impact Factor
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ABSTRACT: The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown. Here, we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drives an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity.
Journal of Molecular Biology 10/2011; 415(1):3-15. · 4.00 Impact Factor
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Xinghua Lou,
Marc Kirchner,
Bernhard Y Renard,
Ullrich Köthe,
Sebastian Boppel,
Christian Graf, Chung-Tien Lee,
Judith A J Steen,
Hanno Steen,
Matthias P Mayer,
Fred A Hamprecht
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ABSTRACT: Time-resolved hydrogen exchange (HX) followed by mass spectrometry (MS) is a key technology for studying protein structure, dynamics and interactions. HX experiments deliver a time-dependent distribution of deuteration levels of peptide sequences of the protein of interest. The robust and complete estimation of this distribution for as many peptide fragments as possible is instrumental to understanding dynamic protein-level HX behavior. Currently, this data interpretation step still is a bottleneck in the overall HX/MS workflow.
We propose HeXicon, a novel algorithmic workflow for automatic deuteration distribution estimation at increased sequence coverage. Based on an L(1)-regularized feature extraction routine, HeXicon extracts the full deuteration distribution, which allows insight into possible bimodal exchange behavior of proteins, rather than just an average deuteration for each time point. Further, it is capable of addressing ill-posed estimation problems, yielding sparse and physically reasonable results. HeXicon makes use of existing peptide sequence information, which is augmented by an inferred list of peptide candidates derived from a known protein sequence. In conjunction with a supervised classification procedure that balances sensitivity and specificity, HeXicon can deliver results with increased sequence coverage.
The entire HeXicon workflow has been implemented in C++ and includes a graphical user interface. It is available at http://hci.iwr.uni-heidelberg.de/software.php.
Supplementary data are available at Bioinformatics online.
Bioinformatics 06/2010; 26(12):1535-41. · 5.47 Impact Factor
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Xinghua Lou,
Marc Kirchner,
Bernhard Y. Renard,
Ullrich Köthe,
Sebastian Boppel,
Christian Graf, Chung-Tien Lee,
Judith A. J. Steen,
Hanno Steen,
Matthias P. Mayer,
Fred A. Hamprecht
Bioinformatics. 01/2010; 26:1535-1541.
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Shinji Tsutsumi,
Mehdi Mollapour,
Christian Graf, Chung-Tien Lee,
Bradley T Scroggins,
Wanping Xu,
Lenka Haslerova,
Martin Hessling,
Anna A Konstantinova,
Jane B Trepel,
Barry Panaretou,
Johannes Buchner,
Matthias P Mayer,
Chrisostomos Prodromou,
Len Neckers
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ABSTRACT: Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes, as it regulates diverse signal transduction nodes that integrate numerous environmental cues to maintain cellular homeostasis. Hsp90 also is secreted from normal and transformed cells and regulates cell motility. Here, we have identified a conserved hydrophobic motif in a beta-strand at the boundary between the N domain and charged linker of Hsp90, whose mutation not only abrogated Hsp90 secretion but also inhibited its function. These Hsp90 mutants lacked chaperone activity in vitro and failed to support yeast viability. Notably, truncation of the charged linker reduced solvent accessibility of this beta-strand and restored chaperone activity to these mutants. These data underscore the importance of beta-strand 8 for Hsp90 function and demonstrate that the functional consequences of weakened hydrophobic contacts in this region are reversed by charged-linker truncation.
Nature Structural & Molecular Biology 11/2009; 16(11):1141-7. · 12.71 Impact Factor
