title: NMR insight into myosin-binding subunit coiled-coil structure reveals binding interface with protein kinase G-Iα leucine zipper in vascular function.
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 ataanddheptad 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.
authors.affiliation: From the Division of Nephrology and Center for Vascular Biology Research, aksharma@bidmc.harvard.edu namasal34@gmail.com.|the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215.
authors.forename: Alok K
authors.initials: AK
authors.lastname: Sharma
authors.affiliation: the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215.|Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Birrane
authors.affiliation: Bruker Biospin Corp., Billerica, Massachusetts 01821, and.
authors.forename: Clemens
authors.initials: C
authors.lastname: Anklin
authors.affiliation: Warp Drive Bio, Inc., Cambridge, Massachusetts 02139.
authors.forename: Alan C
authors.initials: AC
authors.lastname: Rigby
authors.affiliation: From the Division of Nephrology and Center for Vascular Biology Research, salper@bidmc.harvard.edu.|the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215.
authors.forename: Seth L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D016350:Leucine Zippers; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D056004:Molecular Dynamics Simulation; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D009218:Myosins / Q000737:chemistry*; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity
chemical_list: D062965:Cyclic GMP-Dependent Protein Kinase Type I; D009218:Myosins
keywords: coiled-coil; heterotetrameric dimer of dimers; molecular docking; molecular dynamics simulations; myosin; nuclear magnetic resonance (NMR); paramagnetic relaxation enhancement; protein kinase G (PKG); vascular smooth-muscle cells