Use of phosphotyrosine-containing peptides to target SH2 domains: Antagonist peptides of the Crk/CrkL-p130Cas axis.

Autor:	Douglas JT; Nuclear Magnetic Resonance Core Lab, University of Kansas, Lawrence, KS, United States., Johnson DK; Computational Chemical Biology Core, Molecular Graphics and Modeling Laboratory, University of Kansas, Lawrence, Kansas, United States., Roy A; High Throughput Screening Laboratory, University of Kansas, Lawrence, KS, United States., Park T; Department of Pediatrics, Children's Mercy Kansas City and University of Missouri Kansas City School of Medicine, Kansas City, MO, United States.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2024; Vol. 698, pp. 301-342. Date of Electronic Publication: 2024 Apr 27.
DOI:	10.1016/bs.mie.2024.04.013
Abstrakt:	Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention. (Copyright © 2024. Published by Elsevier Inc.)
Databáze:	MEDLINE
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