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

Abstract

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.

Structural modification of NADPH oxidase activator (Noxa 1) by oxidative stress: An experimental and computational study

“…These regions with dynamic mobility may play a role in SH3 domain mediated protein-protein interaction networks [9]. Furthermore, we have compared our Noxa1 SH3 structure with other reported SH3 homologues, i.e., the PDB 5K28 structure [50]. A small difference was observed in RMSD values between Noxa1 SH3 and 5K28, as shown in Table 2.…”

Phage display derived peptides for Alzheimer's disease therapy and diagnosis

“…In 2005, Scholle et al . generated 22 different phage peptide libraries ranging from 8-20 amino acids in length with sizes of up to 10 11 clones, and have used these libraries to select peptide ligands to several protein targets 30 , 31 . Excepted homemade libraries, commercial random peptide libraries and commercial vectors, such as PhD series phage libraries from New England Biolabs, are available to accelerate the work of scientific researchers 32 .…”

Phage Display Selection, Identification, and Characterization of Novel Pancreatic Cancer Targeting Peptides

“…Of these, ovarian-cancer-targeting peptide J18 was reported to exhibit an IC 50 value of 10.5 ± 1.1 µM (mean±std) and was effectively used in imaging of xenografted ovarian tumors in mice [17]. In addition, peptide MIP that was selected to bind Src homology 3 (SH3) domain of human mixed-lineage kinase 3 (MLK3) displayed an IC 50 value of 15.8 ± 0.4 µM (mean±std) [53]. Further, a peptide targeting the breast cancer biomarker cysteine-rich intestinal protein 1 (CRIP1) showed an IC 50 value of 8.8 µM [54].…”