Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation

Sha, Fern; Kurosawa, Kohei; Glasser, Eliezra; Ketavarapu, Gayatri; Albazzaz, Samara; Koide, Akiko; Koide, Shohei

doi:10.1016/j.jmb.2023.168010

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation

Journal Article · Thu Feb 16 00:00:00 EST 2023 · Journal of Molecular Biology

DOI:https://doi.org/10.1016/j.jmb.2023.168010· OSTI ID:2423528

Sha, Fern ^[1]; Kurosawa, Kohei ^[2]; Glasser, Eliezra ^[3]; Ketavarapu, Gayatri ^[3]; Albazzaz, Samara ^[1]; Koide, Akiko ^[4]; Koide, Shohei ^[4]

The University of Chicago, IL (United States)
The University of Chicago, IL (United States); New York University Langone Health, New York, NY (United States)
New York University Langone Health, New York, NY (United States)
The University of Chicago, IL (United States); New York University Langone Health, New York, NY (United States); New York University Grossman School of Medicine, New York, NY (United States)

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the “PTP-dead” C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

View Accepted Manuscript (DOE)

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

Sponsoring Organization:: Cancer Center Support Grant; National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2423528

Journal Information:: Journal of Molecular Biology, Journal Name: Journal of Molecular Biology Journal Issue: 8 Vol. 435; ISSN 0022-2836

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
allostery
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molecular biology
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Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation

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