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

Shen K, Ramirez B, Mapes B, Shen GR, Gokhale V, Brown ME, Santarsiero B, Ishii Y, Dudek SM, Wang T, Garcia JG - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, Illinois, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE characterization of 15N-labeled N-terminal segments of nmMLCK1.(A) Five 15N-labeled 1-494aa proteins (ca. 3 μg each). (B) Two 15N-labeled 1-264aa proteins (ca. 3 μg each).
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pone.0130515.g002: SDS-PAGE characterization of 15N-labeled N-terminal segments of nmMLCK1.(A) Five 15N-labeled 1-494aa proteins (ca. 3 μg each). (B) Two 15N-labeled 1-264aa proteins (ca. 3 μg each).

Mentions: Extending earlier success in nmMLCK protein expression [8, 36, 37], we established a novel bacterial system for structure-oriented protein expression utilizing bacterial IMPACT system (NEB) using a C-terminal intein-chitin-binding domain (CBD) tag, which has an advantage in that the tag can be rendered traceless, i.e., readily cleavable in the presence of free thiols in the late stage of purification [28, 29]. The nmMLCK-encoding MYLK gene was codon-optimized by commercial gene synthesis service (Genscript, NJ) for enhanced bacterial protein expression under low temperature to promote correct protein folding and avoid aggregation. This strategy was successfully used to express and purify multiple wild type and SNP variants of the N-terminal segments of nmMLCK, unlabeled or 15N-labeled in two lengths, which include the 1-494aa segments and 1-264aa segments as verified by SDS-PAGE (Fig 2A and 2B) and MALDI-time of flight (TOF) MS.


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

Shen K, Ramirez B, Mapes B, Shen GR, Gokhale V, Brown ME, Santarsiero B, Ishii Y, Dudek SM, Wang T, Garcia JG - PLoS ONE (2015)

SDS-PAGE characterization of 15N-labeled N-terminal segments of nmMLCK1.(A) Five 15N-labeled 1-494aa proteins (ca. 3 μg each). (B) Two 15N-labeled 1-264aa proteins (ca. 3 μg each).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482139&req=5

pone.0130515.g002: SDS-PAGE characterization of 15N-labeled N-terminal segments of nmMLCK1.(A) Five 15N-labeled 1-494aa proteins (ca. 3 μg each). (B) Two 15N-labeled 1-264aa proteins (ca. 3 μg each).
Mentions: Extending earlier success in nmMLCK protein expression [8, 36, 37], we established a novel bacterial system for structure-oriented protein expression utilizing bacterial IMPACT system (NEB) using a C-terminal intein-chitin-binding domain (CBD) tag, which has an advantage in that the tag can be rendered traceless, i.e., readily cleavable in the presence of free thiols in the late stage of purification [28, 29]. The nmMLCK-encoding MYLK gene was codon-optimized by commercial gene synthesis service (Genscript, NJ) for enhanced bacterial protein expression under low temperature to promote correct protein folding and avoid aggregation. This strategy was successfully used to express and purify multiple wild type and SNP variants of the N-terminal segments of nmMLCK, unlabeled or 15N-labeled in two lengths, which include the 1-494aa segments and 1-264aa segments as verified by SDS-PAGE (Fig 2A and 2B) and MALDI-time of flight (TOF) MS.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, Illinois, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus