Characterization of a novel weak interaction between MUC1 and Src-SH3 using nuclear magnetic resonance spectroscopy

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• 作者：Nirosha Gunasekara^a ; Brian Sykes^b ; ^{brian.sykes@ualberta.ca} ; Judith Hugh^a ; ^{judithh@ualberta.ca}
• 关键词：ICAM-1 ; intercellular adhesion molecule-1 ; CD ; cytoplasmic domain ; SH3 ; Src homology-3 ; NMR ; nuclear magnetic resonance ; Kd ; dissociation constant ; 1H&ndash ; 15N HSQC NMR ; two-dimensional NMR spectra correlating the amide proton and nitrogen NMR chemical s
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 期刊代码：159_0006291x
• 类别：bio
• 出版时间：18 May, 2012
• 卷：421
• 期：4
• 页码：832-836
• 文件大小：525 K

摘要

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 ¹⁵N 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.