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

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

doi:10.1074/jbc.m117.781260

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.

Highlights

The interaction between myosin-binding subunit (MBS)/MYPT1 and protein kinase G (PKG)-I␣ is currently modeled between the N-terminal leucine zipper (LZ) domain of PKG-I␣ and the C-terminal MBSCT180 domain of MLC phosphatase (MLCP)
Our results presented below describe the solution NMR structure of coiled-coil domain (CC) MBS and define at the molecular level the individual amino acid residues and dimer-dimer interface involved in the CC MBS1⁄7LZ PKG-I␣ complex of vascular smooth-muscle cells (VSMC)
Size-exclusion chromatography (SEC) shows that the purified CC MBS polypeptide adopts a dimeric conformation in solution (Fig. 1B; extrapolated dimer mass of 13,275 Da from the calibration curve agrees well with theoretical mass of 13,142.2 Da). 13C/ 15N- or 15N-labeled CC MBS polypeptides of Ͼ96% apparent purity migrated as a single homodimeric peak on SEC (Fig. 1B), consistent with previous results [16]

Summary

Introduction

CGMP activates PKG-I␣, which binds, phosphorylates, and activates MBS of MLCP to promote VSMC relaxation [11]. An MBS domain lacking its CC failed to bind PKG-I␣ [9] These studies taken together suggest the importance of both CC and LZ domains of MBS in formation of the PKG-I␣1⁄7MBS complex. These results provide no atomic scale insight into the binding mechanism, due to the lack of 3D structural information for the subdomains of MBSCT180

Methods

Results

Conclusion