Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism.
The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.
Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, Würzburg, Germany.
Andrea
A
Schulze
Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, Würzburg, Germany.
Gerti
G
Beliu
Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, Würzburg, Germany.
Dominic A
DA
Helmerich
Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, Würzburg, Germany.
Jonathan
J
Schubert
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK.
Laurence H
0000-0002-6910-1809
LH
Pearl
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton, UK.
Chrisostomos
C
Prodromou
Department of Biotechnology and Biophysics, Julius-Maximilians-University Würzburg, Am Hubland, Würzburg, Germany.
Hannes
H
Neuweiler
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D004579:Electron Transport; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009068:Movement; D011487:Protein Conformation; D000072760:Single Molecule Imaging; D015003:Yeasts
D005456:Fluorescent Dyes; D018841:HSP90 Heat-Shock Proteins; D000251:Adenosine Triphosphatases