NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function

Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G...

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Bibliographic Details
Published in:The Journal of biological chemistry 2017-04, Vol.292 (17), p.7052-7065
Main Authors: Sharma, Alok K., Birrane, Gabriel, Anklin, Clemens, Rigby, Alan C., Alper, Seth L.
Format: Article
Language:English
Subjects:
Animals
Circular Dichroism
coiled-coil
Cyclic GMP-Dependent Protein Kinase Type I - chemistry
heterotetrameric dimer of dimers
Hydrophobic and Hydrophilic Interactions
Leucine Zippers
Magnetic Resonance Spectroscopy
Mice
molecular docking
Molecular Dynamics Simulation
molecular dynamics simulations
Muscle, Smooth, Vascular - cytology
myosin
Myosins - chemistry
nuclear magnetic resonance (NMR)
paramagnetic relaxation enhancement
Protein Binding
Protein Domains
protein kinase G (PKG)
Protein Multimerization
Protein Structure and Folding
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Spectrophotometry, Ultraviolet
Static Electricity
vascular smooth-muscle cells
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Summary:Nitrovasodilators relax vascular smooth-muscle cells in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or the leucine zipper (LZ) domain of the myosin light-chain phosphatase component, myosin-binding subunit (MBS), with the N-terminal LZ domain of protein kinase G (PKG)-Iα. Despite the importance of vasodilation in cardiovascular homeostasis and therapy, our structural understanding of the MBS CC interaction with LZ PKG-1α has remained limited. Here, we report the 3D NMR solution structure of homodimeric CC MBS in which amino acids 932–967 form a coiled-coil of two monomeric α-helices in parallel orientation. We found that the structure is stabilized by non-covalent interactions, with dominant contributions from hydrophobic residues at a and d heptad positions. Using NMR chemical-shift perturbation (CSP) analysis, we identified a subset of hydrophobic and charged residues of CC MBS (localized within and adjacent to the C-terminal region) contributing to the dimer-dimer interaction interface between homodimeric CC MBS and homodimeric LZ PKG-Iα. 15N backbone relaxation NMR revealed the dynamic features of the CC MBS interface residues identified by NMR CSP. Paramagnetic relaxation enhancement- and CSP-NMR-guided HADDOCK modeling of the dimer-dimer interface of the heterotetrameric complex exhibits the involvement of non-covalent intermolecular interactions that are localized within and adjacent to the C-terminal regions of each homodimer. These results deepen our understanding of the binding restraints of this CC MBS·LZ PKG-Iα low-affinity heterotetrameric complex and allow reevaluation of the role(s) of myosin light-chain phosphatase partner polypeptides in regulation of vascular smooth-muscle cell contractility.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M117.781260