The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90

Abstract

The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP‐driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N‐terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD−MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD−MD arrangement with the accessibility of a client binding site on the NTD.

Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

“…Phosphate groups in the CL may form salt bridges with the numerous lysine residues in proximity and rigidify the CL, while dephosphorylation may result in increased flexibility. This mechanism could regulate contacts between β-strand 8 and the surrounding secondary structures: α-helix 8, β-strand 2 and in particular β-strand 9, as β-strands 8 and 9 are separated by the CL but do interact in the closed conformations of Hsp90, forming a β-hairpin like structure [ 29 , 31 , 110 , 111 ]. These contacts may then modulate the exposure of local protein binding sites, Hsp90β secretion and global conformational dynamics.…”

“…An early study suggested that the CL in chicken Hsp90 forms two α-helices separated by a proline-containing loop [ 30 ]. A study partly supported this finding: in yeast Hsp90, two regions with residual secondary structure (RSS) separated by a proline containing loop were detected [ 31 ]. The region with RSS at the C-terminal side of the loop, which includes the corresponding β-strand 9 in HtpG, can interact with and increase the exposure of β-strand 8, which serves as a contact site for some Hsp90 clients such as p53.…”

“…Specific features in the CL sequence are known to regulate Hsp90 function and global conformation, but the exact residues exerting these effects have not been determined [ 19 , 20 , 31 ]. The CL in yeast contains four prolines, but only one is conserved in chicken and humans, in the flexible middle loop.…”

Effect of Polyphosphorylation on Behavior of Protein Disordered Regions

“…The charged linker comprising a disordered PASK-motif connects N-terminal domain, which contains ATPbinding site, and middle domain. Since this prolonged disordered linker plays a key role in HSP90 machinery during closing/opening cycle [17,27], polyphosphorylation of the charged linker might be important for the HSP90 functioning. Indeed, polyphosphorylation has been demonstrated to reduce interaction of the charged linker with key structural elements of globular part of the N-terminal domains, including N-terminal helix, and might indirectly influence the behavior of the ATP-binding pocket lid.…”

“…All eukaryotic HSP90 members (including human endoplasmin), in contrast to bacterial (such as HtpG from E. coli) and mitochondrial homologs, contain prolonged disordered N-and C-ends as well as a prolonged charged linker in the C-terminal region of the N-terminal domain (Figure 1B,C). This inter-domain charged linker (287-327 a.a. in human endoplasmin), which is rich in acidic residues and lysine residues and represents a so-called PASK-motif, plays an important role in structural rearrangement of domains during opening/closing cycle and interaction with client proteins [17]; it was earlier shown to be polyphosphorylated. On the contrary, bacterial HSP90 members have only a short beta-hairpin with few lysine residues (Figure 1D).…”