title: Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State.
abstract: GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.
authors.affiliation: From the Department of Chemistry, University of California, Davis, California 95616 and.
authors.forename: Sunghyuk
authors.initials: S
authors.lastname: Lim
authors.affiliation: Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Peshenko
authors.affiliation: Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Olshevskaya
authors.affiliation: Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Dizhoor
authors.affiliation: From the Department of Chemistry, University of California, Davis, California 95616 and jbames@ucdavis.edu.
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D005136:Eye Proteins / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D054878:Lipoylation; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D058767:Protein Unfolding; D011956:Receptors, Cell Surface / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D005136:Eye Proteins; D051604:Guanylate Cyclase-Activating Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C110715:guanylate cyclase 1; D019814:Myristic Acid; D006162:Guanylate Cyclase; D008274:Magnesium; D002118:Calcium
keywords: calcium; calcium-binding protein; calorimetry; guanylate cyclase (guanylyl cyclase); nuclear magnetic resonance (NMR); phototransduction; vision