Limits...
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

Comparison of the HSQC spectra of 15N-labeled 1-264aa/ 1-494aa segments of nmMLCK1.(A) and (B), the HSQC spectra of 1-264aa segments of nmMLCK1: (A) wild type; (B) P147 SNP variant. (C) to (F), the superimposition of the HSQC spectra (the 1st set of spectra shown in blue color and the 2nd in red color): (C) wild type, 1-494aa and 1-264aa; (D) P147, 1-494aa and 1-264aa; (E) 1-264aa, wild type and 147P; (F) zoom in of the squared region in (E). The HSQC spectra of the 1-264aa segments exhibited a better dispersion of 1H-15N chemical shifts, with less degenerate, better resolved signals than 1-494aa segments. Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments demonstrated that the spectra of 1-264aa segments are a recapitulation of the subsets of those of their corresponding 1-494aa segments, suggesting that the shorter segments each possess a structure similar to the corresponding part of their longer counterparts. The same characteristic pattern of signal changes observed for 1-494aa segments is recapitulated with better resolution by superimposition of the spectra of the 1-264aa 147P SNP mutant onto those of the 1-264aa wild type segment, suggesting that the same structural difference exist for the 1-264aa wild type and the P147 SNP mutant.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482139&req=5

pone.0130515.g004: Comparison of the HSQC spectra of 15N-labeled 1-264aa/ 1-494aa segments of nmMLCK1.(A) and (B), the HSQC spectra of 1-264aa segments of nmMLCK1: (A) wild type; (B) P147 SNP variant. (C) to (F), the superimposition of the HSQC spectra (the 1st set of spectra shown in blue color and the 2nd in red color): (C) wild type, 1-494aa and 1-264aa; (D) P147, 1-494aa and 1-264aa; (E) 1-264aa, wild type and 147P; (F) zoom in of the squared region in (E). The HSQC spectra of the 1-264aa segments exhibited a better dispersion of 1H-15N chemical shifts, with less degenerate, better resolved signals than 1-494aa segments. Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments demonstrated that the spectra of 1-264aa segments are a recapitulation of the subsets of those of their corresponding 1-494aa segments, suggesting that the shorter segments each possess a structure similar to the corresponding part of their longer counterparts. The same characteristic pattern of signal changes observed for 1-494aa segments is recapitulated with better resolution by superimposition of the spectra of the 1-264aa 147P SNP mutant onto those of the 1-264aa wild type segment, suggesting that the same structural difference exist for the 1-264aa wild type and the P147 SNP mutant.

Mentions: While a significant portion of the signals in the HSQC spectra of the 1-494aa segments were well dispersed, these segments were less amenable to NMR-based structural analysis due to hard-to-resolve degenerate signals in the region of 8.0–8.6 ppm in 1H chemical shift. Shorter segments of the 1-264aa, the wild type and the P147 SNP mutant, were next examined by HSQC, with the spectra of the two smaller proteins exhibiting excellent dispersion of 1H-15N chemical shifts, with improved signals (Fig 4A and 4B). Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments (Fig 4C and 4D) demonstrated that the spectra of a 1-264aa segment is a recapitulation of the subsets of those of their corresponding 1-494aa segment, suggesting that the shorter segment possesses structural similarity to the longer segment. The same characteristic pattern of signal changes as observed for 1-494aa segments (Fig 3A) is recapitulated with better resolution by superimposition of the spectra of the 1-264aa P147 SNP mutant onto those of the 1-264aa wild type segment (Fig 4E and 4F), suggesting that the same structural difference exist for the 1-264aa wild type and the 147P SNP mutant, as that of the corresponding 1-494aa segments. On the other hand, an otherwise almost identical majority of HSQC signals of the two SNP variants precludes the possibility of a global structural change across the 1-264aa segments and again indicate that any such structural change would be very minor or minimal.


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)

Comparison of the HSQC spectra of 15N-labeled 1-264aa/ 1-494aa segments of nmMLCK1.(A) and (B), the HSQC spectra of 1-264aa segments of nmMLCK1: (A) wild type; (B) P147 SNP variant. (C) to (F), the superimposition of the HSQC spectra (the 1st set of spectra shown in blue color and the 2nd in red color): (C) wild type, 1-494aa and 1-264aa; (D) P147, 1-494aa and 1-264aa; (E) 1-264aa, wild type and 147P; (F) zoom in of the squared region in (E). The HSQC spectra of the 1-264aa segments exhibited a better dispersion of 1H-15N chemical shifts, with less degenerate, better resolved signals than 1-494aa segments. Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments demonstrated that the spectra of 1-264aa segments are a recapitulation of the subsets of those of their corresponding 1-494aa segments, suggesting that the shorter segments each possess a structure similar to the corresponding part of their longer counterparts. The same characteristic pattern of signal changes observed for 1-494aa segments is recapitulated with better resolution by superimposition of the spectra of the 1-264aa 147P SNP mutant onto those of the 1-264aa wild type segment, suggesting that the same structural difference exist for the 1-264aa wild type and the P147 SNP mutant.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130515.g004: Comparison of the HSQC spectra of 15N-labeled 1-264aa/ 1-494aa segments of nmMLCK1.(A) and (B), the HSQC spectra of 1-264aa segments of nmMLCK1: (A) wild type; (B) P147 SNP variant. (C) to (F), the superimposition of the HSQC spectra (the 1st set of spectra shown in blue color and the 2nd in red color): (C) wild type, 1-494aa and 1-264aa; (D) P147, 1-494aa and 1-264aa; (E) 1-264aa, wild type and 147P; (F) zoom in of the squared region in (E). The HSQC spectra of the 1-264aa segments exhibited a better dispersion of 1H-15N chemical shifts, with less degenerate, better resolved signals than 1-494aa segments. Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments demonstrated that the spectra of 1-264aa segments are a recapitulation of the subsets of those of their corresponding 1-494aa segments, suggesting that the shorter segments each possess a structure similar to the corresponding part of their longer counterparts. The same characteristic pattern of signal changes observed for 1-494aa segments is recapitulated with better resolution by superimposition of the spectra of the 1-264aa 147P SNP mutant onto those of the 1-264aa wild type segment, suggesting that the same structural difference exist for the 1-264aa wild type and the P147 SNP mutant.
Mentions: While a significant portion of the signals in the HSQC spectra of the 1-494aa segments were well dispersed, these segments were less amenable to NMR-based structural analysis due to hard-to-resolve degenerate signals in the region of 8.0–8.6 ppm in 1H chemical shift. Shorter segments of the 1-264aa, the wild type and the P147 SNP mutant, were next examined by HSQC, with the spectra of the two smaller proteins exhibiting excellent dispersion of 1H-15N chemical shifts, with improved signals (Fig 4A and 4B). Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments (Fig 4C and 4D) demonstrated that the spectra of a 1-264aa segment is a recapitulation of the subsets of those of their corresponding 1-494aa segment, suggesting that the shorter segment possesses structural similarity to the longer segment. The same characteristic pattern of signal changes as observed for 1-494aa segments (Fig 3A) is recapitulated with better resolution by superimposition of the spectra of the 1-264aa P147 SNP mutant onto those of the 1-264aa wild type segment (Fig 4E and 4F), suggesting that the same structural difference exist for the 1-264aa wild type and the 147P SNP mutant, as that of the corresponding 1-494aa segments. On the other hand, an otherwise almost identical majority of HSQC signals of the two SNP variants precludes the possibility of a global structural change across the 1-264aa segments and again indicate that any such structural change would be very minor or minimal.

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