4.4 Article

Effects of Ca2+, Mg2+, and Myristoylation on Guanylyl Cyclase Activating Protein 1 Structure and Stability

Journal

BIOCHEMISTRY
Volume 48, Issue 5, Pages 850-862

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/bi801897p

Keywords

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Funding

  1. NCRR NIH HHS [S10 RR011973] Funding Source: Medline
  2. NEI NIH HHS [R01 EY012347, EY012347, R01 EY012347-12] Funding Source: Medline
  3. NINDS NIH HHS [R01 NS045909, NS045909] Funding Source: Medline

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Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca2+-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca2+ and Mg2+ 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 Ca2+ at EF3 and EF4 (Delta H-EF3 = -3.5 kcal/mol, and Delta H-EF4 = -0.9 kcal/mol) with nanomolar affinity (K-EF3 = 80 nM, and K-EF4 = 200 nM), and a third Ca2+ binds entropically at EF2 (Delta H-EF2 = 3.1 kcal/mol, and K-EF2 = 0.9,M). GCAP1 binds functionally to Mg2+ at EF2 (Delta H-EF2 = 4.3 kcal/mol, and K-EF2 = 0.7 MM) required for RetGC activation. Call and/or Mg2+ 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 Ca2+, 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 Mg2+ or Ca2+ binding but instead is constitutively dynamic and may play a role in promoting interactions of GCAP1 with the cyclase.

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