Spatially and kinetically resolved changes in the conformational dynamics of the Hsp90 chaperone machine.
The highly conserved 90 kDa heat shock protein (Hsp90) chaperones use ATP to regulate the stability and activity of many signalling molecules like protein kinases and transcription factors. Studies using crystallography, electron microscopy and small-angle X-ray scattering yielded controversial results for the conformational states that these dimeric multidomain proteins assume while progressing through the ATPase cycle. To better understand the molecular mechanism of Hsp90 proteins, we studied the conformational dynamics of the Escherichia coli homologue HtpG in solution using amide hydrogen exchange mass spectrometry (HX-MS) and fluorescence spectroscopy. A conformation-sensitive fluorescent probe allowed to elucidate the ATPase cycle of HtpG. Continuous-labelling and pulse-labelling HX-MS experiments revealed major ATP-induced conformational changes throughout the protein that do not occur simultaneously, but progress surprisingly slow from the immediate nucleotide-binding site towards the N terminus and the middle domain. The conversion between the different conformational states is rate limiting for ATP hydrolysis, and the nucleotide-coordinating residue, Glu34, is important for the rate constant of conversion. Our findings, for the first time, allow to kinetically resolve changes in the conformational dynamics of individual structural elements of Hsp90.
Graf
Christian
C
Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany.
Stankiewicz
Marta
M
Kramer
Günter
G
Mayer
Matthias P
MP
D000255:Adenosine Triphosphate / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018841:HSP90 Heat-Shock Proteins; C489352:HtpG protein, E coli; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate