Molecular chaperones confer stress resistance critical for survival under harsh environmental conditions and maintain protein homeostasis under normal conditions. Beyond their role in protein folding, chaperones affect protein activation and trafficking, facilitating the degradation of terminally misfolded proteins, and the formation and disassembly of macromolecular complexes. Hsp90 is a highly conserved member of the chaperone family, and plays a unique role by its regulatory influence in eukaryotes via the activation of specific classes of substrates (also known as clients), such as nuclear receptors and kinases 1
. This broad regulatory influence is thought to underlie the potent influence of Hsp90 inhibitors on the growth of diverse cancer types 2
. Despite its fundamental cell biological and clinical importance, the mechanism by which Hsp90 stabilizes and remodels client proteins is not understood.
One confounding problem is that Hsp90 is large, conformationally dynamic, and undergoes dramatic structural changes upon ATP binding and hydrolysis ()3; 4; 5
. Small-angle x-ray scattering (SAXS) and electron microscopy measurements (EM) have revealed an underlying complexity of Hsp90’s conformational dynamics 6; 7; 8; 9; 10; 11
. The Hsp90 monomer is composed of three stable domains (N-terminal domain, NTD; middle, MD; C-terminal, CTD), and conformational flexibility results from their rigid body-like rearrangement. Under apo conditions a weak MD/CTD interface allows for a wide range of arm-arm geometries that can be influenced by pH and osmolyte conditions 8; 12
. This striking flexibility has been observed for highly diverse Hsp90 homologs 6; 9
and is postulated to be critically important to Hsp90’s ability to recognize a remarkably diverse set of client proteins.
Hsp90 conformational flexibility
Unlike other molecular chaperones, Hsp90 appears to prefer largely folded but non-native states. This poses an additional practical challenge as such states can be difficult to populate and are prone to aggregation. Previous work introduced a well-behaved model client protein, the partially folded, but non-aggregating protein, Δ131Δ, a fragment of Staphyloccocal Nuclease that has been studied extensively by the protein folding community 13
. Using this model client revealed that (i
) under apo conditions Hsp90 partially closes around Δ131Δ; (ii
) Hsp90 binds a highly structured region of Δ131Δ; (iii
) Δ131Δ accelerates a nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90, effectively activating the chaperone by lowering a rate-limiting conformational barrier. Taken in the context that the ligand-binding domain of GR enhances the ATPase of the human Hsp90 14
and that the ribosomal subunit L2 enhances the ATPase of the bacterial Hsp90 15
, this suggests that activation of the rate-limiting Hsp90 conformational transition is a conserved feature of bone fide
Hsp90 clients, similar to Hsp70 activation by peptide substrates. However, the mechanism by which substrate binding can drive the dramatic Hsp90 open-closed transition is unknown. Indeed, a previous low resolution SAXS analysis 13
could not determine whether Δ131Δ makes cross-monomer contacts as has been observed for the activating cochaperone aha1 16
, or solely intramonomer contacts as observed for an Hsp90-cdc37-cdk4 (chaperone-cochaperone-kinase substrate) EM reconstruction17
The Hsp90 ATPase is slow, on the order of 0.1–1 hydrolysis events per minute depending on the homolog and conditions 18; 19; 20
, and mirrors a slow conformational change from the open apo state to the closed ATP conformation 13; 21
. This dramatic transition involves a large change in arm-arm proximity, a domain-level change in the NTD orientation, and local structural changes within the NTD (lid closure over the nucleotide binding pocket, strand exchange between NTDs) and the MD (restructuring of the catalytic loop) 3; 5; 19; 22
. Although the relative importance of these structural changes to the closure rate is not known, the structures of the AMPPNP-bound canine Grp94 (the Hsp90 homolog specific to the ER) and the apo bacterial Hsp90 (HtpG) suggest that the NTD rotational state plays an important role. Both structures exhibit an open resting state in which the NTDs are diametrically opposed, requiring a significant conformational change to come into a closure-competent alignment 4; 5; 23
. As illustrated in , the required movement involves a 90° rotation and a 25 Å translation of the NTD center of mass, rearranging ~2000 Å2
at the MD interface. This aligns closed-state contacts (, red and blue spheres) and also repositions ATP by ~20 Å allowing the γphosphate to contact a highly conserved arginine on the MD (R336 in HtpG) that is essential for both closure and the bound ATP hydrolysis 3; 20; 24
(). Importantly, a full lid closure over the nucleotide pocket, which appears to be necessary for closure 25
, cannot occur in the NTD resting state due to a significant steric clash with the middle domain 4
. These observations suggest that an NTD rotation may be involved in the timing and order of many critical steps in closure and subsequent ATP hydrolysis.
Here we use our HtpG activating substrate to interrogate the open/closed transition and how this process is substrate-catalyzed. Key questions include (i) whether substrate contacts are within a single monomer or across monomers, (ii) defining the substrate binding region on HtpG in greater detail than could be achieved from our previous SAXS analysis, (iii) establishing whether a single set or multiple substrate contacts are utilized, (iv) determining how substrate binding affects HtpG structural dynamics, particularly at the NTD, and how this is related to the large energetic barrier to closure.