title: Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.
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.
authors.affiliation: Department of Basic Sciences and Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania 19027, USA.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Peshenko
authors.forename: Elena V
authors.initials: EV
authors.lastname: Olshevskaya
authors.forename: Sunghyuk
authors.initials: S
authors.lastname: Lim
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Dizhoor
mesh_terms: D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D006152:Cyclic GMP / Q000378:metabolism; D020832:EF Hand Motifs; D004533:Egtazic Acid / Q000737:chemistry; D006162:Guanylate Cyclase / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry*; D006801:Humans; D008027:Light; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D012160:Retina / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014785:Vision, Ocular
chemical_list: D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; C496737:Guca1a protein, mouse; D004533:Egtazic Acid; D014364:Tryptophan; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium