Structural and functional validation of a highly specific Smurf2 inhibitor

Tessier, Tanner M.; Chowdhury, Arvid; Stekel, Zane; Fux, Julia; Sartori, Maria Augusta; Teyra, Joan; Jarvik, Nick; Chung, Jacky; Kurinov, Igor; Sicheri, Frank; Sidhu, Sachdev S.; Singer, Alex U.; Zhang, Wei

doi:10.1002/pro.4885

Structural and functional validation of a highly specific Smurf2 inhibitor

Journal Article · Tue Dec 26 00:00:00 EST 2023 · Protein Science

DOI:https://doi.org/10.1002/pro.4885· OSTI ID:2470156

^[1]; Chowdhury, Arvid ^[2]; Stekel, Zane ^[1]; Fux, Julia ^[1]; Sartori, Maria Augusta ^[2]; ^[3]; Jarvik, Nick ^[4]; Chung, Jacky ^[4]; Kurinov, Igor ^[5]; Sicheri, Frank ^[6]; Sidhu, Sachdev S. ^[4]; Singer, Alex U. ^[4]; Zhang, Wei ^[1]

Univ. of Guelph, ON (Canada)
Univ. of Toronto, ON (Canada)
Icosagen (Estonia)
Univ. of Waterloo, ON (Canada)
Argonne National Laboratory (ANL), Argonne, IL (United States); Cornell Univ., Ithaca, NY (United States). Northeastern Collaborative Access Team (NECAT)
Mount Sinai Hospital, Toronto, ON (Canada)

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor‐β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross‐reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein‐based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2–E3 protein interaction interface.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2470156

Journal Information:: Protein Science, Journal Name: Protein Science Journal Issue: 2 Vol. 33; ISSN 0961-8368

Publisher:: Wiley -- The Protein SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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crystal structure
inhibitor
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ubiquitin variants

Structural and functional validation of a highly specific Smurf2 inhibitor

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