Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.
Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca(2+) binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a function of Ca(2+) concentration. No Ca(2+)-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca(2+) concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca(2+) dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.
Sokal
I
I
Department of Ophthalmology, University of Washington, Seattle, Washington 98195, USA.
Li
N
N
Klug
C S
CS
Filipek
S
S
Hubbell
W L
WL
Baehr
W
W
Palczewski
K
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D020832:EF Hand Motifs; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005123:Eye / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000737:chemistry; D051604:Guanylate Cyclase-Activating Proteins; D008698:Mesylates / Q000494:pharmacology; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010069:Oxadiazoles / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013455:Sulfur / Q000737:chemistry
D002135:Calcium-Binding Proteins; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D051604:Guanylate Cyclase-Activating Proteins; D008698:Mesylates; D010069:Oxadiazoles; D013113:Spin Labels; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D013455:Sulfur; C034248:(1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate; D006162:Guanylate Cyclase; D003545:Cysteine; D002118:Calcium