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

By Alok K. Sharma, Gabriel Birrane, Clemens Anklin, Alan C. Rigby, Seth L. Alper

July 28, 2017

  • Year

  • Technique

  • Industry

Title

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

Author

Alok K. Sharma, Gabriel Birrane, Clemens Anklin, Alan C. Rigby, Seth L. Alper

Year

2017

Journal

The Journal of Biological Chemistry

Abstract

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.

Full Article

http://www.jbc.org/content/292/17/7052.short

Instrument

J-815

Keywords

Circular dichroism, Secondary structure, Biochemistry