Probing the interaction between the coiled coil leucine zipper of cGMP-dependent protein kinase Ialpha and the C terminus of the myosin binding subunit of the myosin light chain phosphatase.
Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.
Sharma
Alok K
AK
Divison of Molecular and Vascular Medicine, Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Zhou
Guo-Ping
GP
Kupferman
Joseph
J
Surks
Howard K
HK
Christensen
Eva N
EN
Chou
James J
JJ
Mendelsohn
Michael E
ME
Rigby
Alan C
AC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D016350:Leucine Zippers; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D003432:Cross-Linking Reagents; D018994:Myosin Light Chains; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D009218:Myosins; D007930:Leucine
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