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 at and 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. N 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.

The Transmembrane Conformation of the Influenza B Virus M2 Protein in Lipid Bilayers

“…A secondary shift of 0.5 ppm corresponds to a difference in hydrogen-bond length (R N…O ) of ~0.2 Å 59 , which is consistent with the variation in R N…O in the GCN4 crystal structure 51 . These periodic H N secondary shifts are also seen in other coiled coil proteins such as the antiparallel bovine IF 1 60 and the parallel myosin-binding subunit 61 .…”

Karsztos víztartók sérülékenységi térképezési megközelítései a Gömör-Tornai-karszt példáján

“…Ezt a jelkiszélesedést más coiled coil rendszerek NMR spektroszkópiával megvalósuló vizsgálata során is megfigyelték már több alkalommal (Renzoni 2001, Ishigaki 2007, Vavra 2016, sőt kutatókkal való személyes beszélgetések arra engednek következtetni, hogy a publikációkban leírt példáknál többször is előfordul ez a jelenség. Az említett jelkiszélesedés nem minden esetben tapasztalható, így egyes kisebb méretű coiled coil rendszerek jól tanulmányozhatók NMR spektroszkópiával -például a legtöbbet vizsgált coiled coil, a leucincipzár GCN4 vagy más egyéb esetekben (Pascual 1996, Cukier 2016, Gill 2016, Kaplan 2017, Sharma 2017.…”