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

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.

• Authors: Kui Shen, Benjamin Ramirez, Brandon Mapes, Grace R Shen, Vijay Gokhale, Mary E Brown, Bernard Santarsiero, Yoshitaka Ishii, Steven M Dudek, Ting Wang, Joe G N Garcia
• Journal: PloS one (PLoS One) Vol. 10 Issue 6 Pg. e0130515 ( 2015) ISSN: 1932-6203 [Electronic] United States
• PMID: 26111161 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Protein Isoforms
• Myosin-Light-Chain Kinase
• non-muscle myosin light-chain kinase, human

Topics

• Black People (genetics)
• Humans
• Lung Diseases (genetics, pathology)
• Magnetic Resonance Spectroscopy
• Models, Molecular
• Myosin-Light-Chain Kinase (chemistry, genetics, metabolism)
• Nuclear Magnetic Resonance, Biomolecular
• Phosphorylation
• Polymorphism, Single Nucleotide (genetics)
• Protein Isoforms (chemistry, genetics)
• Structure-Activity Relationship