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Igor V. Peshenko, Elena V. Olshevskaya, Sunghyuk Lim, James B. Ames, Alexander M. Dizhoor; The N-fatty Acyl Group In A Bovine Guanylyl Cyclase Activating Protein-1 Provides Intramolecular Tuning Of Its Calcium Sensitivity And Interaction With The Effector Enzyme. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5583.
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© ARVO (1962-2015); The Authors (2016-present)
Guanylyl cyclase activating protein 1 (GCAP1), N-myristoylated neuronal calcium sensor protein, regulates retinal guanylyl cyclase (RetGC) in response to light-dependent changes in free calcium concentrations in photoreceptors. We studied how the interactions between myristoyl group and protein surrounding affected functional properties of GCAP1.
Mutations were introduced to the hydrophobic pocket formed by the parts of EF-hand 1 and 2 and the C-terminal alpha-helix in order to modulate interactions of the N-myristoyl residue with the surrounding amino acid side chains. We tested the stoichiometry of Ca2+ binding, Ca2+-sensitive activation of RetGC1, and the intrinsic tryptophan fluorescence of the mutated GCAP1 in the presence and in the absence of the N-myristoyl group. The position of the myristoyl moiety inside the hydrophobic pocket of GCAP1 between different functional states was verified by NMR-spectroscopy.
The absence of myristoyl moiety did not unfold GCAP1, but decreased Ca2+ sensitivity of GCAP1, its apparent affinity for RetGC, and the maximal level of cyclase activation. The myristoyl residue surrouned by EF hands 1 and 2 critically affected Ca2+ binding in the opposite part of the molecule formed by EF hands 3 and 4. According to NMR spectra, myristoyl remained constrained inside the EF1/EF2 semi-globule both in the Ca2+-bound (RetGC inhibitor) and Mg2+-bound (RetGC activator) states in L80F/L176F/V180F mutant. This mutant displayed much higher than wild type affinity for the cyclase, but much lower than the wild type sensitivity to Ca2+. The Phe176 drastically increased the apparent affinity of GCAP1 for the cyclase and also altered Ca2+-sensitivity of the RetGC regulation by shifting it to a higher than normal range of free Ca2+. In comparison with mutations at the mid-portion of the fatty acyl group, substitutions at the bottom and the top of the hydrophobic cavity affected the regulatory properties of GCAP1 to a lesser extent.
The fatty acyl group in GCAP1, through its interaction with the C-terminal helix that bridges the N- and the C-proximal portions of the molecule, creates a dynamic tug that tunes GCAP1 into the optimal conformation as a cyclase regulator, by balancing the effectiveness of the interaction with the target enzyme with the optimal Ca2+ sensor function.
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