Recoverin is a Ca2؉ -regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca 2؉ sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca 2؉ sensitivity.Many biological processes are triggered or regulated by transient intracellular Ca 2ϩ signals. Because these signals elicit specific cellular responses, the precise detection of changes in cytoplasmic Ca 2ϩ concentration is a crucial step in many signaling pathways and requires sensing of Ca 2ϩ within very different concentration ranges. Ca 2ϩ -binding proteins work as intracellular Ca 2ϩ sensors and regulate their targets with high specificity and high spatial and temporal resolution. To achieve these remarkable tasks, Ca 2ϩ is recognized by specific amino acid sequence motifs, for example the C 2 domain and the EF-hand motif (1, 2). These motifs can detect subtle changes in Ca 2ϩ concentration and allow a fine tuning of Ca 2ϩ signaling. However, it remains a challenging problem to understand at a structural level how minimal changes in cytoplasmic Ca 2ϩ are reliably detected.The EF-hand superfamily of Ca 2ϩ -binding proteins includes, among others, the family of neuronal calcium sensor (NCS) 3 proteins (3), which are named because of their predominant expression in neuronal tissue. NCS proteins are grouped into five subfamilies and show a rather heterogeneous localization and function in the nervous system (4). In the photoreceptor cells of the vertebrate retina, for instance, recoverin and several isoforms of guanylate cyclase activating protein (GCAP) detect changes in Ca 2ϩ concentration during or after illumination and regulate their target proteins in Ca 2ϩ -dependent feedback loops (5).Recoverin inhibits rhodopsin kinase at high cytoplasmic Ca 2ϩ concentration (6 -9), a process that is thought to contribute to light adaptation of photoreceptor cells (9, 10). Recoverin harbors a myristoyl group at its N terminus (11), which is buried in a hydrophobic cleft in the Ca 2ϩ -free state (12). Upon Ca 2ϩ binding to the two functional EF-hands (EF-hand 2 and EFhand 3) (13) the acyl chain is exposed to the solvent. This socalled Ca 2ϩ -myrist...

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Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn2+ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 106 M-1) and one low-affinity (2 × 105 M-1) Zn2+-binding sites, whereas Mg2+-loaded and Ca2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn2+-binding sites of apo- and Mg2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn2+ concentrations displace Mg2+ from the EF-hands and decrease the stoichiometry of Ca2+ binding. Meanwhile, one of the EF-hands of Zn2+-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn2+ binding to Ca2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca2+-free NCS-1 and favours the interaction of its Ca2+-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.

Recoverin belongs to the family of intracellular Ca(2+)-binding proteins containing EF-hand domains, neuronal calcium sensors (NCS). In photoreceptor outer segments, recoverin is involved into the recovery of visual cycle via Ca(2+)-dependent interaction with disk membranes and inhibition of rhodopsin kinase. The function of a conservative within NCS family Cys residue in the inactive EF-loop 1 remains unclear, but previous study has shown its vulnerability to oxidation under mild oxidizing conditions. To elucidate the influence of oxidation of the conservative Cys39 in recoverin the properties of its C39D mutant, mimicking oxidative conversion of Cys39 into sulfenic, sulfinic or sulfonic acids have been studied using intrinsic fluorescence, circular dichroism, and equilibrium centrifugation methods. The C39D substitution results in essential changes in structural, physico-chemical and physiological properties of the protein: it reduces α-helical content, decreases thermal stability and suppresses protein affinity for photoreceptor membranes. The latter effect precludes proper functioning of the Ca(2+)-myristoyl switch in recoverin. The revealed significance of oxidation state of Cys39 for maintaining the protein functional status shows that it may serve as redox sensor in vision and suggests an explanation of the available data on localization and light-dependent translocation of recoverin in rod photoreceptors.