Guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins myristoylated at the N terminus that regulate guanylate cyclases in photoreceptor cells and belong to the family of neuronal calcium sensors (NCS). Many NCS proteins display a recoverin-like "calcium-myristoyl switch" whereby the myristoyl group, buried inside the protein in the Ca(2+)-free state, becomes fully exposed upon Ca(2+) binding. Here we present a 2.0 A resolution crystal structure of myristoylated GCAP1 with Ca(2+) bound. The acyl group is buried inside Ca(2+)-bound GCAP1. This is in sharp contrast to Ca(2+)-bound recoverin, where the myristoyl group is solvent exposed. Furthermore, we provide direct evidence that the acyl group in GCAP1 remains buried in the Ca(2+)-free state and does not undergo switching. A pronounced kink in the C-terminal helix and the presence of the myristoyl group allow clustering of sequence elements crucial for GCAP1 activity.

Photon absorption by rhodopsin triggers the phototransduction signaling pathway that culminates in degradation of cGMP, closure of cGMP-gated ion channels and hyperpolarization of the photoreceptor membrane. This process is accompanied by a decrease in free Ca 2+ concentration in the photoreceptor cytosol sensed by Ca 2+ -binding proteins that modulate phototransduction and activate the recovery phase to reestablish the photoreceptor dark potential. Guanylate cyclase-activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) family and are responsible for activating retinal guanylate cyclases (retGCs) at low Ca 2+ concentrations triggering synthesis of cGMP and recovery of the dark potential. Here we review recent structural insight into the role of the N-terminal myristoylation in GCAPs and compare it to other NCS family members. We discuss previous studies identifying regions of GCAPs important for retGC1 regulation in the context of the new structural data available for myristoylated GCAP1. In addition, we present a hypothetical model for the Ca 2+ -triggered conformational change in GCAPs and retGC1 regulation. Finally, we briefly discuss the involvement of mutant GCAP1 proteins in the etiology of retinal degeneration as well as the importance of other Ca 2+ sensors in the modulation of phototransduction.The phototransduction pathway links absorption of light to a decrease in cytosolic cGMP. Depletion of the cGMP pool induces closure of cGMP-gated cation channels and hyperpolarization of photoreceptor cells with a consequent decrease in glutamate neurotransmitter release detected by secondary neurons (1). Briefly, phototransduction is initiated with absorption of a photon by the chromophore 11-cis-retinal that is covalently linked to G-protein-coupled receptors known as opsins. Isomerization of 11-cis-retinal to all-trans-retinal and dissociation of the chromophore produces a conformational change in the opsin and consequent activation of the coupled heterotrimeric G-protein transducin (2). Activated transducin then activates a retina-specific phosphodiesterase (PDE6) that cleaves cGMP, depleting cytoplasmic cGMP and closing cGMP-gated cation channels.

SummaryAbsorption of light by visual pigments initiates the phototransduction pathway that results in degradation of the intracellular pool of cyclic-GMP (cGMP). This hydrolysis promotes the closing of cGMP-gated cation channels and consequent hyperpolarization of rod and cone photoreceptor cell membranes. Guanylate Cyclase Activating Proteins (GCAPs) are a family of proteins that regulate retinal guanylate cyclase (GC) activity in a Ca 2+ -dependent manner. At high [Ca 2+ ], typical of the dark-adapted state (~500 nM), GCAPs inhibit retinal GCs. At the low [Ca 2+ ] (~50 nM) that occur after the closing of cGMP-gated channels, GCAPs activate retinal GCs to replenish dark-state cGMP levels. Here, we report the crystal structure of unmyristoylated human GCAP3 with Ca 2+ bound. GCAP3 is an EF-hand Ca 2+ -binding protein with Ca 2+ bound to EF2, 3 and 4, while Ca 2+ binding to EF-hand 1 is disabled. GCAP3 contains two domains with the EF-hand motifs arranged in a tandem array similar to GCAP2 and members of the recoverin subfamily of Ca 2+ -binding proteins. Residues not involved in Ca 2+ binding, but conserved in all GCAPs, cluster around EF1 in the N-terminal domain and may represent the interface with GCs. Five point mutations in the closely related GCAP1 have been linked to the etiology of cone dystrophies. These residues are conserved in GCAP3 and the structure suggests important roles for these amino acids. We present a homology model of GCAP1 based on GCAP3 that offers insight into the molecular mechanism underlying the autosomal dominant cone dystrophies produced by GCAP1 mutations.

We report herein the NMR structure of Tm0979, a structural proteomics target from Thermotoga maritima. The Tm0979 fold consists of four ␤/␣ units, which form a central parallel ␤-sheet with strand order 1234. The first three helices pack toward one face of the sheet and the fourth helix packs against the other face. The protein forms a dimer by adjacent parallel packing of the fourth helices sandwiched between the two ␤-sheets. This fold is very interesting from several points of view. First, it represents the first structure determination for the DsrH family of conserved hypothetical proteins, which are involved in oxidation of intracellular sulfur but have no defined molecular function. Based on structure and sequence analysis, possible functions are discussed. Second, the fold of Tm0979 most closely resembles YchN-like folds; however the proteins that adopt these folds differ in secondary structural elements and quaternary structure. Comparison of these proteins provides insight into possible mechanisms of evolution of quaternary structure through a simple mechanism of hydrophobicity-changing mutations of one or two residues. Third, the Tm0979 fold is found to be similar to flavodoxin-like folds and ␤/␣ barrel proteins, and may provide a link between these very abundant folds and putative ancestral half-barrel proteins.

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