title: Characterization of a novel weak interaction between MUC1 and Src-SH3 using nuclear magnetic resonance spectroscopy.
abstract: Breast cancer causes death through cancer cell migration and subsequent metastasis to distant organs. In vitro, the MUC1 mucin can mediate breast cancer cell migration by binding to intercellular adhesion molecule-1 (ICAM-1). This migration is dependent on MUC1 cytoplasmic domain (MUC1-CD) activation of the non-receptor tyrosine kinase, Src, possibly through competitive displacement of an inhibitory Src intramolecular SH3 binding. Therefore, we characterized the binding site and affinity of the MUC1-CD for Src-SH3 using multidimensional nuclear magnetic resonance (NMR) spectroscopy to monitor the titration of the (15)N labeled Src-SH3 domain with synthetic native and mutant peptides of MUC1-CD. The results revealed that the dissociation constant (K(d)) for the interaction of the native MUC1-CD peptides and Src-SH3 domain was weak with a K(d) of 2-3 mM. Notably, the SH3 residues most perturbed upon peptide binding were located outside the usual hydrophobic binding cleft in a previously described alternate binding site on the Src-SH3, suggesting that MUC1-CD binds to a non-canonical site. The binding characteristics outlined here suggest that the interaction between Src-SH3 and MUC1-CD represents a novel weak electrostatic interaction of the type which is increasingly recognized as important in transient and dynamic protein complexes required for cell migration and signal transduction. As such, this study forms the foundation for the design of specific inhibitors of this interaction which may target breast cancer metastases with exquisite specificity.
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, 5B4.21 WCM Health Science Centre, 8440-112th Street, Edmonton, Alberta, Canada T6G 2R7.
authors.forename: Nirosha
authors.initials: N
authors.lastname: Gunasekara
authors.forename: Brian
authors.initials: B
authors.lastname: Sykes
authors.forename: Judith
authors.initials: J
authors.lastname: Hugh
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002465:Cell Movement; D005260:Female; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry*; D060066:Protein Interaction Maps; D017433:Protein Structure, Secondary; D055672:Static Electricity; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry*
chemical_list: C060192:MUC1 protein, human; D018396:Mucin-1; D019061:src-Family Kinases