Heat shock protein 90’s mechanochemical cycle is dominated by thermal fluctuations

Abstract

The molecular chaperone and heat shock protein 90 (Hsp90) exists mainly as a homodimer in the cytoplasm. Each monomer has an ATPase in its N-terminal domain and undergoes large conformational changes during Hsp90's mechanochemical cycle. The threecolor single-molecule assay and data analysis presented in the following allows one to observe at the same time nucleotide binding and the conformational changes in Hsp90. Surprisingly, and completely unlike the prior investigated systems, nucleotides can bind to the N-terminally open and closed state without strictly forcing the protein into a specific conformation. Both the transitions between the conformational states and the nucleotide binding/ unbinding are mainly thermally driven. Furthermore, the two ATP binding sites show negative cooperativity; i.e., nucleotides do not bind independently to the two monomers. We thus reveal a picture of how nucleotide binding and conformational changes are connected in the molecular chaperone Hsp90, which has far-ranging consequences for its function and is distinct from previously investigated motor proteins.conformational dynamics | three-color FRET | single molecule | intramolecular communication | protein structure M olecular chaperones are proteins that assist the folding or unfolding and the assembly or disassembly of other proteins (1). A very abundant molecular chaperone in eukaryotes is the heat shock protein 90 (Hsp90) comprising around 1% of cytosolic protein (2, 3). The protein consists of two elongated monomers that have two dimerization interfaces in the N domain and in the C domain, respectively. Like motor proteins, it is an ATPase, but the mechanistic function of ATP binding and hydrolysis is still unclear. Current models assume that one ATP each binds to the N-terminal domains in the open state, followed by N-terminal closing and ATP hydrolysis in a strict succession (4)-similar to all to-date investigated motor proteins (5-9). Because of experimental limitations, it was not possible to directly observe nucleotide binding and conformational changes at the same time and thus detect the mechanochemical cycle of Hsp90. To overcome this limitation we designed a three color single-molecule Förster resonance energy transfer (smFRET) assay with alternating laser excitation (ALEX). This allows one to detect when ATP binds to which monomer and at the same time to read out the conformational state of Hsp90. Surprisingly, we find that ATP binds to the N-terminal open and close conformations, but not independently. Therefore, our data show that the picture of a successive conformational cycle has to be replaced by stochastic transitions in-between conformations of Hsp90. Nevertheless, there is an anticorrelated communication between the N termini throughout the whole protein.

Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

“…Therefore, our results support previous findings showing that asymmetry plays an important role in homodimeric Hsp90 systems: asymmetry is required for client binding 39 , asymmetric SUMOylation facilitates cochaperone binding 40 , nucleotide affinity is asymmetric 22 and hydrolysis is only required in one subunit 41,42 .…”

“…The cycle for the N-terminal open-close movement has recently been solved 22 . Putting the numbers from this publication into equation (1) we obtain a 'directionality' of around 1 k B T. This is the thermal energy and therefore consistent with the finding, that idle Hsp90 is mainly dominated by thermal fluctuations.…”

“…As in our previous studies, we used the yeast Hsp90 C-terminal Zipper constructs yHsp90 61C and yHsp90 385C for the single molecule experiments and the same constructs without zipper for the ensemble experiments (Figs 2a, b, 3a). Heterologous expression was done in E. coli and purification was done via Ni NTA, ion exchange and size exclusion chromatography 22 .…”

“…Hsp90 is a dimer consisting of two elongated chains lying in parallel; each chain contains an amino-terminal (N-terminal) ATP binding site [14][15][16] . Large N-terminal openclose movements of the dimer occur in all investigated Hsp90 systems [17][18][19][20][21][22][23][24][25][26] . Likely, these open-close movements are stronger coupled to ATP hydrolysis in the bacterial Hsp90 (HtpG) compared with the eukaryotic yeast Hsp90, which might raise the question why Hsp90 lost some nucleotide control during evolution from prokaryotes to eukaryotes.…”

“…First we determine suitable positions for the fluorophores in the multicomponent system consisting of a yeast Hsp90 dimer, yeast p23 (Sba1) and ATP. The mapping for Hsp90's conformational changes and nucleotide binding has been done before 22 , and resulted in labelling of ATP at the g-phosphate and the two Hsp90 monomers at amino acid position 385 in the M-domain and position 61 in the N-domain (Fig. 2d).…”