RESUMO
Phototransduction in vertebrate photoreceptor cells is controlled by Ca2+-dependent feedback loops involving the membrane-bound guanylate cyclase GC-E that synthesizes the second messenger guanosine-3',5'-cyclic monophosphate. Intracellular Ca2+-sensor proteins named guanylate cyclase-activating proteins (GCAPs) regulate the activity of GC-E by switching from a Ca2+-bound inhibiting state to a Ca2+-free/Mg2+-bound activating state. The gene GUCY2D encodes for human GC-E, and mutations in GUCY2D are often associated with an imbalance of Ca2+ and cGMP homeostasis causing retinal disorders. Here, we investigate the Ca2+-dependent inhibition of the constitutively active GC-E mutant V902L. The inhibition is not mediated by GCAP variants but by Ca2+ replacing Mg2+ in the catalytic center. Distant from the cyclase catalytic domain is an α-helical domain containing a highly conserved helix-turn-helix motif. Mutating the critical amino acid position 804 from leucine to proline left the principal activation mechanism intact but resulted in a lower level of catalytic efficiency. Our experimental analysis of amino acid positions in two distant GC-E domains implied an allosteric communication pathway connecting the α-helical and the cyclase catalytic domains. A computational connectivity analysis unveiled critical differences between wildtype GC-E and the mutant V902L in the allosteric network of critical amino acid positions.
RESUMO
Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3', 5'-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca2+-sensor proteins, named guanylate-cyclase-activating proteins. A rotation model of activation involving an α-helix rotation was described as a common activation motif among hormone-receptor GCs. We tested whether the photoreceptor GC-E underwent an α-helix rotation when reaching the active state. We experimentally simulated such a transitory switch by integrating alanine residues close to the transmembrane region, and compared the effects of alanine integration with the point mutation V902L in GC-E. The V902L mutation is found in patients suffering from retinal cone-rod dystrophies, and leads to a constitutively active state of GC-E. We analyzed the enzymatic catalytic parameters of wild-type and mutant GC-E. Our data showed no involvement of an α-helix rotation when reaching the active state, indicating a difference in hormone receptor GCs. To characterize the protein conformations that represent the transition to the active state, we investigated the protein dynamics by using a computational approach based on all-atom molecular dynamics simulations. We detected a swinging movement of the dimerization domain in the V902L mutant as the critical conformational switch in the cyclase going from the low to high activity state.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase , Guanilato Ciclase , Alanina/metabolismo , Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/química , Hormônios/metabolismo , Humanos , Células Fotorreceptoras Retinianas Cones/metabolismoRESUMO
Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca2+ and confer Ca2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg2+-bound, Ca2+-free, Fe2+-free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca2+-bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe2+-dependent regulation of cyclase activity in zebrafish photoreceptors.