title: Effects of Ca2+, Mg2+, and myristoylation on guanylyl cyclase activating protein 1 structure and stability.
abstract: Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca(2+)-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca(2+) and Mg(2+) binding to the individual EF-hands, characterize metal-induced conformational changes, and evaluate structural effects of myristoylation as studied by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). GCAP1 binds cooperatively to Ca(2+) at EF3 and EF4 (DeltaH(EF3) = -3.5 kcal/mol, and DeltaH(EF4) = -0.9 kcal/mol) with nanomolar affinity (K(EF3) = 80 nM, and K(EF4) = 200 nM), and a third Ca(2+) binds entropically at EF2 (DeltaH(EF2) = 3.1 kcal/mol, and K(EF2) = 0.9 microM). GCAP1 binds functionally to Mg(2+) at EF2 (DeltaH(EF2) = 4.3 kcal/mol, and K(EF2) = 0.7 mM) required for RetGC activation. Ca(2+) and/or Mg(2+) binding to GCAP1 dramatically alters DSC and NMR spectra, indicating metal-induced protein conformational changes in EF2, EF3, and EF4. Myristoylation of GCAP1 does not significantly alter its metal binding energetics or NMR spectra, suggesting that myristoylation does not influence the structure of the metal-binding EF-hands. Myristoylation also has almost no effect on protein folding stability measured by DSC. NMR resonances of myristate attached to GCAP1 are exchange-broadened, upfield-shifted, and insensitive to Ca(2+), consistent with the myristoyl group being sequestered inside the protein as seen in the crystal structure. We conclude that the protein environment near the myristate is not influenced by Mg(2+) or Ca(2+) binding but instead is constitutively dynamic and may play a role in promoting interactions of GCAP1 with the cyclase.
authors.affiliation: Department of Chemistry, University of California, Davis, California 95616, USA.
authors.forename: Sunghyuk
authors.initials: S
authors.lastname: Lim
authors.forename: Igor
authors.initials: I
authors.lastname: Peshenko
authors.forename: Alexander
authors.initials: A
authors.lastname: Dizhoor
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins; D019814:Myristic Acid; D006162:Guanylate Cyclase; D008274:Magnesium; D002118:Calcium