The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function

Abstract

The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N-and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 k B T (2.7 kJ/mol) through binding of the CL to the Ndomain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance. T he molecular chaperone Hsp90 (heat shock protein 90) is essential for the folding, maturation, and activation of approximately 10% of the yeast proteome. The set of substrate proteins is structurally and functionally diverse and ranges from telomerase to kinases and transcription factors (1-3). Processing of these substrates requires ATP turnover and large conformational rearrangements within Hsp90 (4-6). Interestingly, these conformational states of yeast Hsp90 are not strictly coupled to the binding of nucleotides (7) but are recognized and regulated by the interaction with cochaperones (8) and substrate proteins (9).Hsp90 is a dimer with each monomer consisting of three domains (N, M, and C). The N-terminal domain comprises the nucleotide binding site, whereas the M-domain is important for the binding of many substrates. The C-terminal domain is mainly responsible for the dimerization of the protein. A long charged linker (CL) region, amino acids 211-272 in yeast, connects the N-and M-domain in eukaryotes (Fig. 1A). The crystal structure of yeast Hsp90 was obtained by partly deleting the CL region and shows a closed, compact conformation in the presence of AMP-PNP [Adenosine 5′-(β,γ-imido)triphosphate] and Sba1/p23 (10). The fact that the CL region is difficult to map structurally led to the assumption that this region is disordered and flexible, thereby enabling the conformational rearrangements of Hsp90 (11,12). Besides the structural indetermination of the CL, its ultimate function remains elusive as well (13). Early studies show that parts of the CL region (amino acids 211-259) are dispensable in yeast (14), whereas more extended deletions affect cell viability, substrate maturation, and regulation by cochaperones (11). These deficiencies can be partially rescued by a short linker consisting of an artificial sequence (11), but its specific amino acid sequence is associated with a gain of function, probably an additional Hsp90 regulatory mechanism (15).Single-molecule experiments have recently provided detailed insight int...

Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

“…1B shows three stretch and relax cycles obtained at a slow pulling velocity of 10 nm/s in which a single Hsp90 monomer was consecutively unfolded and refolded. The unfolding traces (gray) show a characteristic unfolding pattern exhibiting three major peaks that we had previously identified as the unfolding of the C, N, and M domains of Hsp90 (13). At low forces before the domains unfold, fast transitions can be observed that arise from the rapid docking and undocking of the charged linker that connects the N and M domains ("#" in Fig.…”

“…At low forces before the domains unfold, fast transitions can be observed that arise from the rapid docking and undocking of the charged linker that connects the N and M domains ("#" in Fig. 1B) (13). The completely unfolded Hsp90 monomer starts refolding (purple traces) at high forces (∼5 pN) through a complex sequence of near-equilibrium transitions and folding intermediates.…”

“…These DNA oligonucleotides are then hybridized with long (545 base pair) DNA handles by a single-stranded overhang on one end that is complementary to the DNA oligonucleotides (9). At the other end, DNA handles are functionalized with biotin or digoxigenin, which in turn can bind to streptavidin-coated or anti-digoxigenin-coated 1-μm silica beads (9,13). For trapping of beads, we use a custom-built, dualtrap optical tweezers setup with back-focal plane detection and high resolution as described previously (31).…”

“…All experiments are performed in a buffer containing 40 mM Hepes, 150 mM KCl, 10 mM MgCl 2 , pH 7.4. To avoid photo damage, a scavenger system comprising glucose, glucose oxidase, and glucose catalase or trolox is used (13). A detailed description of methods used is given in SI Methods.…”

“…Hsp90 consists of three domains, the N-terminal domain (N domain, 211 residues), the middle domain (M domain, 266 residues), and the C-terminal domain (C domain, 172 residues). In eukaryotic Hsp90, the N and M domains are connected by a long (62 residues) charged linker that can bind transiently to the N domain (13). In addition, the C domain is partly unstructured (residues 678-709).…”