title: 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.
abstract: 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.
authors.affiliation: 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.
authors.forename: Alok K
authors.initials: AK
authors.lastname: Sharma
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhou
authors.forename: Joseph
authors.initials: J
authors.lastname: Kupferman
authors.forename: Howard K
authors.initials: HK
authors.lastname: Surks
authors.forename: Eva N
authors.initials: EN
authors.lastname: Christensen
authors.forename: James J
authors.initials: JJ
authors.lastname: Chou
authors.forename: Michael E
authors.initials: ME
authors.lastname: Mendelsohn
authors.forename: Alan C
authors.initials: AC
authors.lastname: Rigby
mesh_terms: 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
chemical_list: 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
references.citation: Science. 1993 Nov 26;262(5138):1401-7
references.pmid: 8248779
references.citation: Cell Signal. 2003 Oct;15(10):937-44
references.pmid: 12873707
references.citation: Structure. 2002 Oct;10(10):1349-61
references.pmid: 12377121
references.citation: Protein Sci. 2007 Feb;16(2):323-8
references.pmid: 17189475
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 24;104(17):7062-7
references.pmid: 17438295
references.citation: Biophys J. 2003 Nov;85(5):3279-85
references.pmid: 14581228
references.citation: Nat Med. 2005 Feb;11(2):115-6
references.pmid: 15692588
references.citation: J Biol Chem. 2001 Jun 29;276(26):23895-902
references.pmid: 11312259
references.citation: Biochemistry. 1994 Mar 29;33(12):3515-31
references.pmid: 8142349
references.citation: Biopolymers. 1990;30(7-8):657-76
references.pmid: 2275971
references.citation: Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8259-63
references.pmid: 7667278
references.citation: Science. 1991 May 24;252(5009):1162-4
references.pmid: 2031185
references.citation: J Lipid Res. 2008 May;49(5):1103-12
references.pmid: 18268358
references.citation: Science. 2007 Sep 7;317(5843):1393-7
references.pmid: 17717153
references.citation: Cell. 2003 May 30;113(5):621-30
references.pmid: 12787503
references.citation: Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14545-50
references.pmid: 16195386
references.citation: J Biol Chem. 2004 Jul 30;279(31):32464-73
references.pmid: 15155731
references.citation: J Biomol NMR. 2000 Dec;18(4):319-27
references.pmid: 11200526
references.citation: J Biochem. 2007 Jun;141(6):855-66
references.pmid: 17416594
references.citation: J Clin Invest. 2005 Apr;115(4):840-4
references.pmid: 15841174
references.citation: J Mol Graph. 1996 Feb;14(1):51-5, 29-32
references.pmid: 8744573
references.citation: Biochemistry. 2007 Jan 16;46(2):514-25
references.pmid: 17209561
references.citation: Nat Struct Mol Biol. 2005 Feb;12(2):144-51
references.pmid: 15665873
references.citation: J Biol Chem. 2003 Apr 11;278(15):13462-7
references.pmid: 12556468
references.citation: Circ Res. 2007 Nov 26;101(11):1078-80
references.pmid: 18040024
references.citation: Cell. 1992 Feb 21;68(4):699-708
references.pmid: 1739975
references.citation: J Biomol NMR. 1995 Nov;6(3):277-93
references.pmid: 8520220
references.citation: J Biol Chem. 1992 Feb 5;267(4):2664-70
references.pmid: 1733963
references.citation: Am J Physiol Cell Physiol. 2007 Jan;292(1):C432-9
references.pmid: 16870832
references.citation: Biochemistry. 1995 May 9;34(18):6164-74
references.pmid: 7742321
references.citation: Proc Natl Acad Sci U S A. 2008 May 6;105(18):6702-7
references.pmid: 18448676
references.citation: Chembiochem. 2004 Feb 6;5(2):170-6
references.pmid: 14760737
references.citation: Biochemistry. 1994 Apr 5;33(13):3862-71
references.pmid: 8142389
references.citation: Protein Sci. 2005 Sep;14(9):2421-8
references.pmid: 16131665
references.citation: J Mol Biol. 2007 Nov 9;373(5):1198-212
references.pmid: 17904578
references.citation: Trends Biotechnol. 2006 Jan;24(1):22-7
references.pmid: 16216358
references.citation: J Biol Chem. 2001 Sep 7;276(36):34318-22
references.pmid: 11448958
references.citation: Annu Rev Biophys Biomol Struct. 1998;27:357-406
references.pmid: 9646872
references.citation: Protein Sci. 2007 Nov;16(11):2391-402
references.pmid: 17905831
references.citation: Nat Med. 2003 Dec;9(12):1506-12
references.pmid: 14608379
references.citation: Protein Eng. 1993 Jun;6(4):383-90
references.pmid: 8332596
references.citation: Biochemistry. 1991 Oct 1;30(39):9387-95
references.pmid: 1892839
references.citation: J Mol Biol. 2001 Nov 16;314(1):129-38
references.pmid: 11724538
references.citation: J Mol Biol. 2005 Apr 8;347(4):841-54
references.pmid: 15769474
references.citation: Nat Struct Biol. 2003 Aug;10(8):607-13
references.pmid: 12858163
references.citation: Curr Biol. 1993 Oct 1;3(10):658-67
references.pmid: 15335856
references.citation: Nature. 1994 Nov 17;372(6503):231-6
references.pmid: 7969467
references.citation: J Biol Chem. 2004 Jan 2;279(1):597-603
references.pmid: 14530290
references.citation: Mol Cell Biochem. 1999 Jan;190(1-2):79-84
references.pmid: 10098973
references.citation: Curr Opin Struct Biol. 2000 Oct;10(5):585-92
references.pmid: 11042458
references.citation: Nature. 2000 Mar 9;404(6774):197-201
references.pmid: 10724174
references.citation: FEBS Lett. 1999 Jun 25;453(3):254-9
references.pmid: 10405155
references.citation: Science. 1999 Nov 19;286(5444):1583-7
references.pmid: 10567269