The Three-Dimensional Structure of the cGMP-Dependent Protein Kinase I - α Leucine Zipper Domain and Its Interaction with the Myosin Binding Subunit.

Abstract

Vascular smooth muscle contractile state is tightly coupled to myosin light chain phosphorylation. Myosin light chain phosphorylation is regulated by the counter regulatory activities of myosin light chain kinase and myosin phosphatase. Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase. This in turn functions to dephosphorylate myosin light chains. Mechanistically this involves a protein-protein interaction mediated by a leucine zipper (LZ) domain that is localized within the N-terminus of cGK1α and the C-terminus of myosin-binding subunit (MBS). To better understand the importance of this interaction in regulating smooth muscle vascular tone we have determined the three-dimensional structure of the LZ domain of cGK1α, (cGKIα1–59) using triple resonance, multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have expressed the LZ domain (cGKIα1–59) in E. coli under conditions facilitating the uniform incorporation of 13C/15N and 2H isotopic labels and purified this expressed protein. Circular dichroism data supported that cGK1α is comprised of an α-helix, while our NMR data identified that this helix commences at residue Ala9 and extends through to residue Leu47 of the LZ domain. 15N backbone longitudinal and transverse relaxation rates are best fit to a rotational diffusion anisotropy model that is consistent with a coiled-coil dimmer structure, not a monomeric helix. This suggests that cGKIα1–59 is comprised of a coiled-coil conformation over this same region. A time course investigation carried out with the LZ domain and a homobifunctional amine reactive crosslinker [dithio-bis(succinimidyl propionate)] (DSP) supported the presence of a homodimer. Furthermore, 15N heteronuclear single quantum correlation (HSQC) chemical shift mapping data has been used to identify residues that are critical for interacting with the C-terminal region of the myosin binding subunit (MBS). These data have allowed us to identify those residues necessary for cGK1α dimerization, while simultaneously permitting a better understanding of the critical determinants of cGK1α and MBS that are involved in regulating the contractile state of smooth muscles within blood vessels. The spatial and temporal contractile state of these blood vessels is important in the physiologic control of blood vessel tone and plays a role in the pathogenesis of hypertension and atherosclerosis a leading cause of death in the US and worldwide.