Solution structure of the Hop TPR2A domain and investigation of target druggability by NMR, biochemical and in silico approaches

Abstract

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in tumour biology by promoting the stabilisation and activity of oncogenic ‘client’ proteins. Inhibition of Hsp90 by small-molecule drugs, acting via its ATP hydrolysis site, has shown promise as a molecularly targeted cancer therapy. Owing to the importance of Hop and other tetratricopeptide repeat (TPR)-containing cochaperones in regulating Hsp90 activity, the Hsp90-TPR domain interface is an alternative site for inhibitors, which could result in effects distinct from ATP site binders. The TPR binding site of Hsp90 cochaperones includes a shallow, positively charged groove that poses a significant challenge for druggability. Herein, we report the apo, solution-state structure of Hop TPR2A which enables this target for NMR-based screening approaches. We have designed prototype TPR ligands that mimic key native ‘carboxylate clamp’ interactions between Hsp90 and its TPR cochaperones and show that they block binding between Hop TPR2A and the Hsp90 C-terminal MEEVD peptide. We confirm direct TPR-binding of these ligands by mapping 1H–15N HSQC chemical shift perturbations to our new NMR structure. Our work provides a novel structure, a thorough assessment of druggability and robust screening approaches that may offer a potential route, albeit difficult, to address the chemically challenging nature of the Hop TPR2A target, with relevance to other TPR domain interactors.

The Hsp70–Hsp90 go-between Hop/Stip1/Sti1 is a proteostatic switch and may be a drug target in cancer and neurodegeneration

“…Those types of cancer that rely both on Hsp70–Hsp90-mediated protein folding and proteasome-mediated protein degradation may be particularly vulnerable since the inhibition of Hop reduces the proteasomal activity, which itself is an already established anti-cancer target. Some compounds have been demonstrated to inhibit the interaction between Hsp90 and the TPR2A domain of Hop [ 51 , 187 , 188 ]. The challenge will be to develop inhibitors that are as specific as possible for the interaction of Hop with either Hsp70 or Hsp90, without affecting the interactions of the latter with other TPR-containing co-chaperones.…”

“…Overall, structurally, the TPR domains are highly organized α-helices [ 47 ]. The TPR1, TPR2A, and TPR2B domain structures of yeast and human Sti1/Hop were evaluated either by X-ray diffraction or solution NMR [ 16 , 49 – 51 ]. These studies experimentally confirmed the predicted α-helical structures of the TPR domains of Hop.…”

Optimizing the First TPR Domain of the Human SPAG1 Protein Provides Insight into the HSP70 and HSP90 Binding Properties

“…Interestingly, using combined in silico and biochemical approaches, the TPR2A domain of the protein Hop was turned into a druggable target. 45 Indeed, authors designed an organic ligand that binds the HSP90 recognition site through contacts with the carboxylate-clamp residues. Similarly, Dictyoceratin-A and -C are two inhibitor molecules that bind the TPR1 region of RPAP3.…”

Targeting Chaperone/Co-Chaperone Interactions with Small Molecules: A Novel Approach to Tackle Neurodegenerative Diseases

“…Recently, Darby et al mimicked key native "carboxylate clamp" interactions between Hsp90 and its TPR co-chaperones and designed several compounds that block the interaction between Hop TPR2A and Hsp90 C-terminal MEEVD peptide after the failure of AlphaScreen high-throughput screening [146]. The binding of these compounds to the Hop TPR2A domain was confirmed by mapping 1 H- 15 N HSQC chemical shift perturbations to their new reported NMR solution-state structure of Hop TPR2A.…”