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Abstract
Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity.
Highlights
N-Myristoylated Guanylyl cyclase-activating protein 1 (GCAP1) controls Ca2ϩ sensitivity of cGMP synthesis in photoreceptors
It cannot be ruled out that fatty acylation improves stability of at least some regions in GCAP1 by the embedded fatty chain [24], but we found in the present study that the role of myristoylation is much more than possible general stabilization of protein structure
Stoichiometry of Ca2ϩ Binding by GCAP1 Does Not Depend on Its N-Fatty Acylation—The stoichiometry of Ca2ϩ binding tested by fluorescent indicator dye BAPTA-2 titration [37] was the same for the non-acylated wild-type GCAP1 as for the myristoylated GCAP1 [37]: three Ca2ϩ ions bound to the protein (Fig. 2)
Summary
N-Myristoylated GCAP1 controls Ca2ϩ sensitivity of cGMP synthesis in photoreceptors. Results: Hydrophobic residues replacing the myristoyl group in GCAP1 restore activation of its target enzyme but not Ca2ϩ binding. Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca2؉ sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca2؉ sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca2؉ binding to EF-hand 4. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca2؉-bound and Mg2؉bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca2؉ sensitivity of the cyclase regulation. Protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or “tug”) between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca2؉ sensitivity
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