Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

Kui Shen,Grace R Shen,Vijay Gokhale,Bernard Santarsiero,Ting Wang,Joe G N Garcia,Yoshitaka Ishii,Steven M Dudek,Mary E Brown,Benjamin Ramirez,Brandon Mapes

doi:10.1371/journal.pone.0130515

Kui Shen, Grace R Shen + Show 9 more

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https://doi.org/10.1371/journal.pone.0130515

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Journal: PloS one	Publication Date: Jun 25, 2015
Citations: 61	License type: CC BY 4.0

Affiliation: University of Illinois at Chicago, University of Arizona

Abstract

The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

Highlights

The responses of pulmonary endothelial cells (ECs) to external stimuli, including bioactive agonists and mechanical stress, largely proceed through the spatially-targeted rearrangement of the dynamic, functionally complex actin cytoskeleton [1, 2]
We initially chose a limited number of such proteins for nuclear magnetic resonance (NMR) studies based on our previous studies that P147 SNP variant was more highly associated with acute lung injury (ALI) and asthma as well as on the assumption that 147P/S mutation may be directly involved in changing posttranslational modification
Using NMR and homology modeling, we have defined the potential influence of two MYLK coding SNPs, P147S, on the structure and function of non-muscle isoform of myosin light chain kinase (nmMLCK), as impacting phosphorylation and proteinprotein interaction

Summary

Introduction

The responses of pulmonary endothelial cells (ECs) to external stimuli, including bioactive agonists and mechanical stress, largely proceed through the spatially-targeted rearrangement of the dynamic, functionally complex actin cytoskeleton [1, 2]. NmMLCK is essential to lung vascular barrier protection and recovery induced by barrier-enhancing or barrier-restoring agonists such as hepatocyte growth factor [13] and sphingosine 1-phosphate (S1P) [14] These agonists dramatically drive rapid and spatially-distinct increases in actin polymerization and MLC phosphorylation that are confined to lamellipodial membrane protrusions and the cortical actin ring, resulting in closure of inflammation-mediated paracellular gaps thereby restoring barrier integrity [13,14,15]. This dual role for nmMLCK in critical lung processes highlights the need to understand spatiotemporal regulatory mechanisms for this key multi-functional enzyme

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