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

Research output: Contribution to journalArticle

6 Scopus citations

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 <sup>1</sup>H-<sup>15</sup>N 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.

Original languageEnglish (US)
Article numbere0130515
JournalPLoS One
Volume10
Issue number6
DOIs
StatePublished - Jun 25 2015

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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