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 G Birrane, Clemens Anklin, Alan C Rigby, Seth L. Alper
Year
2017
Journal
The Journal of Biological Chemistry
Abstract
Nitrovasodilators relax vascular smooth muscle cells (VSMC) in part by modulating the interaction of the C-terminal coiled-coil domain (CC) and/or leucine zipper (LZ) domain of the myosin light-chain phosphatase (MLCP) 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-Iα has remained limited. Here, we report the three-dimensional NMR solution structure of homodimeric CC MBS in which aa 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 (PRE) and CSP NMR guided HADDOCK modeling of the dimer-dimer interface of the hetero-tetrameric 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 re-evaluation of the role(s) of MLCP partner polypeptides in regulation of VSMC contractility.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry