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A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin.

ABSTRACT: Recoverin, a 23-kDa Ca(2+)-binding protein of the neuronal calcium sensing (NCS) family, inhibits rhodopsin kinase, a Ser/Thr kinase responsible for termination of photoactivated rhodopsin in rod photoreceptor cells. Recoverin has two functional EF hands and a myristoylated N terminus. The myristoyl chain imparts cooperativity to the Ca(2+)-binding sites through an allosteric mechanism involving a conformational equilibrium between R and T states of the protein. Ca(2+) binds preferentially to the R state; the myristoyl chain binds preferentially to the T state. In the absence of myristoylation, the R state predominates, and consequently, binding of Ca(2+) to the non-myristoylated protein is not cooperative. We show here that a mutation, C39A, of a highly conserved Cys residue among NCS proteins, increases the apparent cooperativity for binding of Ca(2+) to non-myristoylated recoverin. The binding data can be explained by an effect on the T/R equilibrium to favor the T state without affecting the intrinsic binding constants for the two Ca(2+) sites.

SUBMITTER: Ranaghan MJ

PROVIDER: S-EPMC3861663 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin.

Ranaghan Matthew J MJ Kumar Ramasamy P RP Chakrabarti Kalyan S KS Buosi Vanessa V Kern Dorothee D Oprian Daniel D DD

The Journal of biological chemistry 20131104 50

Recoverin, a 23-kDa Ca(2+)-binding protein of the neuronal calcium sensing (NCS) family, inhibits rhodopsin kinase, a Ser/Thr kinase responsible for termination of photoactivated rhodopsin in rod photoreceptor cells. Recoverin has two functional EF hands and a myristoylated N terminus. The myristoyl chain imparts cooperativity to the Ca(2+)-binding sites through an allosteric mechanism involving a conformational equilibrium between R and T states of the protein. Ca(2+) binds preferentially to th ...[more]

PMID: 24189072

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Project description:Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca2+ sensor (NCS) proteins. Among them, the Ca2+ sensor recoverin undergoes a pronounced Ca2+-dependent conformational change, a prototypical so-called Ca2+-myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different recoverin isoforms (zRec1a, zRec1b, zRec2a, and zRec2b) that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca2+-sensitive working modes for zebrafish recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca2+-sensing properties of the different recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular recoverin. Ca2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of recoverin isoforms to changing intracellular Ca2+ during illumination.

Project description:Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1? (IL-1?) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1? production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.

Project description:Neuronal Calcium Sensors (NCS) are highly conserved proteins specifically expressed in neurons. Calcium (Ca2+)-binding to their EF-hand motifs results in a conformational change, which is crucial for the recognition of a specific target and the downstream biological process. Here we present a comprehensive analysis of the allosteric communication between Ca2+-binding sites and the target interfaces of three NCS, namely NCS1, recoverin (Rec), and GCAP1. In particular, Rec was investigated in different Ca2+-loading states and in complex with a peptide from the Rhodopsin Kinase (GRK1) while NCS1 was studied in a Ca2+-loaded state in complex with either the same GRK1 target or a peptide from the D2 Dopamine receptor. A Protein Structure Network (PSN) accounting for persistent non-covalent interactions between amino acids was built for each protein state based on exhaustive Molecular Dynamics simulations. Structural network analysis helped unveiling the role of key amino acids in allosteric mechanisms and their evolutionary conservation among homologous proteins. Results for NCS1 highlighted allosteric inter-domain interactions between Ca2+-binding motifs and residues involved in target recognition. Robust long range, allosteric protein-target interactions were found also in Rec, in particular originating from the EF3 motif. Interestingly, Tyr 86, involved the hydrophobic packing of the N-terminal domain, was found to be a key residue for both intra- and inter-molecular communication with EF3, regardless of the presence of target or Ca2+ ions. Finally, based on a comprehensive topological PSN analysis for Rec, NCS1, and GCAP1 and multiple sequence alignments with homolog proteins, we propose that an evolution-driven correlation may exist between the amino acids mediating the highest number of persistent interactions (high-degree hubs) and their conservation. Such conservation is apparently fundamental for the specific structural dynamics required in signaling events.

Dataset Information

A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin.

Publications

A highly conserved cysteine of neuronal calcium-sensing proteins controls cooperative binding of Ca2+ to recoverin.

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