Abstract
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.
Highlights
Changes in intracellular [Ca2ϩ] play a central role in the regulation of various physiological processes, either by the direct action of Ca2ϩ on effector enzymes, cation channels, and other proteins or, frequently, through processes mediated by Ca2ϩ-binding proteins
In the three-dimensional model of GCAP1, based on the x-ray structure of Ca2ϩbound form of recoverin and the NMR structure of GCAP2 (Fig. 1B), these four Cys are located in different regions of the protein and are ideally suited for reporting local changes upon Ca2ϩ binding
Cys29 and Cys106 are present in the acidic part of GCAP1 containing EF-hand loops, whereas Cys18 and Cys125 are projecting toward the opposite ends of GCAP1 (Fig. 1B)
Summary
GCAP1-His Expression Vector and Site-directed Mutagenesis—The bovine GCAP1 coding sequence was amplified by polymerase chain reaction with primers, which inserted a His tag before the stop codon. The reaction of DTNB with native and modified forms of GCAP1 and its mutants was carried out either in 900 l of guanidine hydrochloride (final concentration, 4 M), pH 8.0, or in 900 l of 10 mM BTP, pH 8.0, containing 100 mM NaCl, 0.4 mM EDTA, and 156.25 M to 390.6 M CaCl2. After 2 h of incubation at room temperature, the sample was ultracentrifuged (10 min at 100,000 ϫ g at 4 °C) and loaded on a Ni2ϩ-nitrilotriacetic acid column equilibrated with 50 mM Tris/HCl, pH 8.0, and 300 mM NaCl. Excess dye was removed by washing the column with the same buffer. GCAP1 mutants modified by IANBD were repurified by Ni2ϩ-nitrilotriacetic acid chromatography as described above. Site-directed Spin Labeling and EPR Spectroscopy—Purified GCAP1 mutants containing single Cys residues (typically ϳ100 M) were incubated with a 3-fold molar excess of the sulfhydryl-specific nitroxide spin label (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate Proteins were detected at 280 nm, 0.4-ml fractions were collected by SDS-PAGE, and an aliquot from each fraction was examined for the GC stimulatory (inhibitory) activity
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