Wydorski, P. M. | Search bioRxiv

bioRxiv2021

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

biophysics↗

bioRxiv2020

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation

Molecular chaperones, including Hsp70/Hsp40 families, play central roles in binding substrates to prevent their aggregation. How Hsp40s select different conformations of substrates remains poorly understood. Here, we report a novel interaction between the Hsp40 DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a {beta}-turn element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau {beta}-turn fragments, but not mutant fragments, can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. This identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

biophysics↗