@article{22d5b0f38a3d41bca8fe9d3848b43e2c,
title = "Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP protein ENAH",
abstract = "The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can{\textquoteright}t be fully understood outside of their native context.",
author = "Theresa Hwang and Parker, {Sara S.} and Hill, {Samantha M.} and Grant, {Robert A.} and Ilunga, {Meucci W.} and Venkatesh Sivaraman and Ghassan Mouneimne and Keating, {Amy E.}",
note = "Funding Information: This project was supported by NIGMS award R01 GM129007 to AEK, NCI award R01 CA196885-01 to GM, and the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. Part of this work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16 M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. TH was partially supported by NIGMS T32 GM007287 and a fellowship from the Koch Institute for Integrative Cancer Research. We thank the Koch Institute's Robert A Swanson (1969) Biotechnology Center for technical support, specifically the Flow Cytometry Core Facility for assistance with fluorescence-activated cell sorting. We thank the MIT Structural Biology Core for assistance with X-ray crystallography, the MIT Biophysical Instrumentation Facility for instrumentation resources, and the MIT BioMicroCenter for assistance with high-throughput sequencing. We thank D Whitney for his contributions to the MassTitr technology, and we thank members of the Keating lab and F Gertler for their thoughtful input. We also thank J Tadros and F Gertler for reagents and M Li, S Elledge, and Brigham and Women?s hospital for the T7-pep library. Funding Information: This project was supported by NIGMS award R01 GM129007 to AEK, NCI award R01 CA196885-01 to GM, and the Koch Institute Support (core) Grant P30-CA14051 from the National Cancer Institute. Part of this work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16 M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. TH was partially supported by NIGMS T32 GM007287 and a fellowship from the Koch Institute for Integrative Cancer Research. Publisher Copyright: {\textcopyright} Hwang et al.",
year = "2022",
month = jan,
doi = "10.7554/eLife.70680",
language = "English (US)",
volume = "11",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
}