One of the Ca2+ binding sites of recoverin exclusively controls interaction with rhodopsin kinase

Recoverin is a neuronal calcium sensor protein that controls the activity of rhodopsin kinase in a Ca(2+)-dependent manner. Mutations in the EF-hand Ca2+ binding sites are valuable tools for investigating the functional properties of recoverin. In the recoverin mutant E121Q (Rec E121Q ) the high-affinity Ca2+ binding site is disabled. The non-myristoylated form of Rec E121Q binds one Ca2+ via its second Ca(2+)-binding site (EF-hand 2), whereas the myristoylated variant does not bind Ca2+ at all. Binding of Ca2+ to non-myristoylated Rec E121Q apparently triggers exposure of apolar side chains, allowing for association with hydrophobic matrices. Likewise, an interaction surface for the recoverin target rhodopsin kinase is constituted upon Ca2+ binding to the non-acylated mutant. Structural changes resulting from Ca(2+)-occupation of EF-hand 2 in myristoylated and non-myristoylated recoverin variants are discussed in terms of critical conditions required for biological activity.     

Recoverin as a redox-sensitive protein

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman's test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells.     

Ca2+-myristoyl switch in the neuronal calcium sensor recoverin requires different functions of Ca2+-binding sites

Recoverin is an EF-hand Ca(2+)-binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca(2+)-dependent manner. It undergoes a Ca(2+)-myristoyl switch when Ca(2+) binds to EF-hand 2 and 3. We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca(2+) binding. EF-hand 2 and 3 display different properties and serve different functions. Binding of Ca(2+) to recoverin is a sequential process, wherein EF-hand 3 is occupied first followed by the filling of EF-hand 2. After EF-hand 3 bound Ca(2+), the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes. In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold. We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.     

Structure and calcium-binding studies of a recoverin mutant (E85Q) in an allosteric intermediate state

Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 microM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45 degrees swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.     

Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.     

Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647

Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. Ca2+-dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide-thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca2+-myristoyl switch of labeled recoverin was monitored by Ca2+-dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca2+-concentration was varied, labeled myristoylated recoverin showed a 37%-change in fluorescence emission and a 34%-change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time-resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca2+-induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant RecE85Q that has a disabled EF-hand 2, but no such influence was detected with the mutant RecE121Q (EF-hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group.     

Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin

Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).     

Binding of ions and hydrophobic probes to alpha-lactalbumin and kappa-casein as determined by analytical affinity chromatography

The technique of analytical affinity chromatography was extended to characterize binding of ions and hydrophobic probes to proteins. Using the immobilized protein mode of chromatography, alpha-lactalbumin and kappa-casein were covalently attached to 200-nm-pore-diameter controlled-pore glass beads and accommodated for high-performance liquid chromatography. The existence of a high affinity binding site (Kdiss = 0.16 microM) (site I) for calcium ion in alpha-lactalbumin was confirmed by chromatography of [45Ca2+]. In addition, chromatography of the hydrophobic probes, 1-(phenylamino)-8-naphthalene-sulfonate (ANS)2 and 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) indicated that Ca2+ bound to a second site (presumably the zinc site or site II) with weaker affinity. Dissociation constants obtained for apo-alpha-lactalbumin were about 80 microM for ANS and 4.7 microM for bis-ANS in the absence of sodium ion. Addition of Ca2+ initially caused a reduction in surface hydrophobicity (lowered affinity for the probe dyes) followed by an increase at higher Ca2+ concentrations (greater than 0.5 mM), suggesting that occupancy of site II restores an apo-like conformation to the protein. Moreover, the effect of Zn2+ was similar to that observed in the higher Ca2+ concentration range, whereas Na+ apparently bound to site I. A calcium binding site of moderate affinity also exists in kappa-casein (Kdiss = 15.6 microM). A cluster of negative charges, probably including the orthophosphate group, most likely comprise this binding site. By preventing self-association, analytical affinity chromatography permits microscale characterization of ligand equilibria in proteins that are unaffected by protein-protein interactions.     

Point amino acid substitutions in the Ca2+-binding centers of recoverin. I. Mechanism of successive filling of Ca2+-binding centers

The molecule of photoreceptor Ca(2+)-binding protein recoverin contains four potential Ca(2+)-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the interaction of Ca2+ with recoverin and its mutant forms containing point amino acid substitutions at the working Ca(2+)-binding sites by measuring the intrinsic protein fluorescence and found that the substitution of Gln for Glu residues chelating Ca2+ in one (the second or the third) or simultaneously in both (the second and the third) Ca(2+)-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (for Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca(2+)-ions. On the other hand, the Gln for Glu85 substitution only in the second site moderately affects its affinity to the cation. Hence, we assumed that recoverin successively binds Ca(2+)-ions: the second site is filled with the cation only after the third site has been filled. The binding constants for the third and the second Ca(2+)-binding sites of recoverin determined by spectrofluorimetric titration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.     

Probing different conformational states of bovine alpha-lactalbumin: fluorescence studies with 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate]

The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bis-ANS) to bovine alpha-lactalbumin (alpha-LA) was investigated. A strong dependence of the Kd value with the bound calcium stoichiometry was found, with Kd values ranging from 6.2 +/- 0.4 to 64.6 +/- 5.9 microM for apo-alpha-LA and 1:1 Ca(II)-alpha-LA, respectively. A 350-fold enhancement of the bis-ANS emission was observed in the protein-bis-ANS complex, along with an approximately 33-nm blue shift. Both appeared to be related to the hydrophobicity of the binding site and were independent of the Ca(II) ion content. From the difference in bis-ANS affinity between apo-alpha-LA and Ca(II)-alpha-LA, we demonstrated that Zn(II) and Al(III) were able to "lock" the protein into a new "apo-like" conformation, which was similar to, but not identical with, the apo conformation. The protein could be interconverted between all three conformations in a Mn(II) titration. The first Mn(II) shifted the apoprotein to the Ca(II) conformation; at higher Mn(II) levels, binding to the second site shifted the protein toward the apo-like conformation. The same behavior was observed with calcium in large excess. The evidence supported a model for the mutually nonexclusive binding of metals both to site I ("calcium site") and to site II ("zinc site") simultaneously. The results suggest that alpha-lactalbumin possesses a hydrophobic surface that becomes somewhat less accessible upon 1:1 calcium binding in the absence of metals that also bind to the zinc site.     

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k)

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.     

Impact of N-terminal myristoylation on the Ca2+-dependent conformational transition in recoverin

Recoverin is a Ca2+-regulated signal transduction modulator found in vertebrate retina that has been shown to undergo dramatic conformational changes upon Ca2+ binding to its two functional EF-hand motifs. To elucidate the differential impact of the N-terminal myristoylation as well as occupation of the two Ca2+ binding sites on recoverin structure and function, we have investigated a non-myristoylated E85Q mutant exhibiting virtually no Ca2+ binding to EF-2. Crystal structures of the mutant protein as well as the non-myristoylated wild-type have been determined. Although the non-myristoylated E85Q mutant does not display any functional activity, its three-dimensional structure in the presence of Ca2+ resembles the myristoylated wild-type with two Ca2+ but is quite dissimilar from the myristoylated E85Q mutant. We conclude that the N-terminal myristoyl modification significantly stabilizes the conformation of the Ca2+-free protein (i.e. the T conformation) during the stepwise transition toward the fully Ca2+-occupied state. On the basis of these observations, a refined model for the role of the myristoyl group as an intrinsic allosteric modulator is proposed.     

Structure,topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers

Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca(2+)-myristoyl switch. Samples of (15)N-labeled Ca(2+)-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of (15)N and (31)P chemical shifts observed in solid-state NMR spectra. On the basis of a (2)H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR ((1)H-(15)N PISEMA) spectra of uniformly and selectively (15)N-labeled recoverin show that the Ca(2+)-bound protein is positioned on the membrane surface such that its long molecular axis is oriented approximately 45 degrees with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.     

Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments

Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca2+ concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca2+ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50=0.76 microm in DRMs and 1.91 microm in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-beta-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca2+-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.     

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.     

Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D(28K)

Calbindin-D(28K) is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D(28K) reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of alpha5 (EF3), alpha8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D(28K) adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D(28K) provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.