Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other...

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Published in:	The Journal of biological chemistry Vol. 293; no. 35; pp. 13553 - 13565
Main Authors:	Kokoszka, Malgorzata E., Kall, Stefanie L., Khosla, Sehar, McGinnis, Jennifer E., Lavie, Arnon, Kay, Brian K.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 31-08-2018 American Society for Biochemistry and Molecular Biology
Subjects:	affinity selection alanine scanning Amino Acid Sequence Binding Sites combinatorial peptides Crystallography, X-Ray Humans ligand-binding protein MAP Kinase Kinase Kinases - chemistry MAP Kinase Kinase Kinases - metabolism Mitogen-Activated Protein Kinase Kinase Kinase 11 mixed-lineage protein kinase MLK3 SH3 domain-interacting peptide Molecular Docking Simulation n-Src insert Peptide Library Peptides - chemistry Peptides - metabolism phage display Protein Binding protein-protein interaction Signal Transduction Src homology 3 domain (SH3 domain) src Homology Domains ligand-binding protein alanine scanning phage display mixed-lineage protein kinase combinatorial peptides protein-protein interaction n-Src insert Src homology 3 domain (SH3 domain) MLK3 SH3 domain-interacting peptide affinity selection
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Summary:	Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated “MLK3 SH3–interacting peptide” (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a β-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1–4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 National Institutes of Health (NIH) Edited by Wolfgang Peti Co-first authors.
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.RA117.000262