Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin

Calretinin-22k (CR-22k) is a splice product of calretinin (CR) found specifically in cancer cells, and possesses four EF-hands and a differently processed C-terminal end. The Ca2+-binding properties of recombinant human calretinin CR-22k were investigated by flow dialysis and spectroscopic methods and compared with those of CR. CR possesses four Ca2+-binding sites with positive cooperativity (nH = 1.3) and a [Ca2+]0.5 of 1.5 microM, plus one low affinity site with an intrinsic dissociation constant (K'D) of 0.5 mM. CR-22k contains three Ca2+-binding sites with nH of 1.3 and [Ca2+]0.5 of 1.2 microM, plus a low affinity site with K'D of 1 mM. All the sites seem to be of the Ca2+-specific type. Limited proteolysis and thiol reactivity suggest that that the C terminus of full-length CR, but not of CR-22k, is in close proximity of site I leading to mutual shielding. Circular dichroism (CD) spectra predict that the content of alpha-helix in CR and CR-22k is similar and that Ca2+ binding leads to very small changes in the CD spectra of both proteins. The optical properties are very similar for CR-22k and CR, even though CR-22k possesses one additional Trp at the C-terminal end, and revealed that the Trp residues are organized into a hydrophobic core in the metal-free proteins and become even better shielded from the aqueous environment upon binding of Ca2+. The fluorescence of the hydrophobic probe 2-p-toluidinylnaphtalene-6-sulfonate is markedly enhanced by the two proteins already in the absence of Ca2+ and is further increased by binding of Ca2+. The trypsinolysis patterns of CR and CR-22k are markedly dependent on the presence or absence of Ca2+. Together, our data suggest the presence of an allosteric conformational unit encompassing sites I-III for CR-22k and I-IV for CR, with a very similar conformation and conformational changes for both proteins. In the allosteric unit of CR, site IV is fully active, whereas in CR-22k this site has a 80-fold decreased affinity, due to the decreased amphiphilic properties of the C-terminal helix of this site. Some very specific Ca2+-dependent conformational changes suggest that both CR and CR-22k belong to the "sensor"-type family of Ca2+-binding proteins.     

Core mutations that promote the calcium-induced allosteric transition of bovine recoverin

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.     

Calcium-dependent and -independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin

The ubiquitous Ca2+-binding regulatory protein calmodulin (CaM) binds and activates a wide range of regulatory enzymes. The binding is usually dependent on the binding of Ca2+ to CaM; however, some target proteins interact with CaM in a calcium-independent manner. In this work, we have studied the interactions between CaM and a 20-residue synthetic peptide encompassing the major calmodulin-binding domain of cyclic nucleotide phosphodiesterase (PDE1A2). The binding was studied in the absence and presence of Ca2+ by far-UV and near-UV circular dichroism, fluorescence, and infrared spectroscopy. In addition, two-dimensional heteronuclear NMR studies with 13C-methyl-Met-CaM and uniformly 15N-labeled CaM were performed. Competition assays with smooth muscle myosin light chain kinase revealed a Kd of 224 nM for peptide binding to Ca2+-CaM, while binding of the peptide to apo-CaM is weaker. The peptide binds with an alpha-helical structure to both lobes of Ca2+-saturated CaM, and the single Trp residue is firmly anchored into the C-terminal lobe of CaM. In contrast, the Trp residue plays a minor role in the binding to the apo-protein. Moreover, when bound to apo-CaM, the PDE peptide is only partially helical, and it interacts solely with the C-terminal lobe of CaM. These results show that the Ca2+-induced activation of PDE involves a significant change in the structure and positioning of the CaM-bound PDE peptide domain.     

Change in conformation of plasma membrane phospholipid scramblase induced by occupancy of its Ca2+ binding site

Phospholipid (PL) scramblase is a 35.1 kDa plasma membrane protein that mediates the accelerated transbilayer migration of plasma membrane PL in activated, injured, or apoptotic cells exposed to elevated intracellular Ca2+. We recently identified a conserved segment in the PL scramblase polypeptide (residues Asp273 to Asp284) that is essential for its PL-mobilizing function and was presumed to contain the Ca2+ binding site of the protein (Zhou, Q., Sims, P. J., and Wiedmer, T. (1998) Biochemistry 37, 2356-2360). Whereas the sequence of this peptide segment resembles that of known Ca2+-binding loops within EF-hand containing proteins, it is unusual in being a single such loop in the entire protein and in being closely spaced to the predicted transmembrane helix (Ala291-Gly309). To gain insight into how Ca2+ activates the PL-mobilizing function of PL scramblase, we analyzed conformational changes associated with occupancy of this putative Ca2+ binding site. In addition to activation by Ca2+, the PL-mobilizing function of PL scramblase was found to be activated by other ions, with apparent affinities Tb3+, La3+ > Ca2+ > Mn2+ > Zn2+ > Sr2+ > Ba2+, Mg2+. Evidence for coordinate binding of metal ion by the polypeptide was provided by resonance energy transfer from protein Trp to Tb3+, which was competed by excess Ca2+. Metal binding to PL scramblase was accompanied by increased right-angle light scattering and by a prominent change in circular dichroism, suggesting that coordinate binding of the metal ion induces a conformational change that includes self-aggregation of the polypeptide. Consistent with this interpretation, addition of Ca2+ was found to protect PL scramblase from proteolysis by trypsin both in detergent solution as well as in situ, within the erythrocyte membrane. Mutation in the segment Asp273-Asp284 reduced Tb3+ incorporation and attenuated the change in CD spectrum induced by bound metal ligand, confirming that this suspected EF-hand loopike segment of the polypeptide directly contributes to the Ca2+ binding site.     

Mutagenesis of the C2 domain of protein kinase C-alpha. Differential roles of Ca2+ ligands and membrane binding residues

The C2 domains of conventional protein kinase C (PKC) have been implicated in their Ca2+-dependent membrane binding. The C2 domain of PKC-alpha contains several Ca2+ ligands that bind multiple Ca2+ ions and other putative membrane binding residues. To understand the roles of individual Ca2+ ligands and protein-bound Ca2+ ions in the membrane binding and activation of PKC-alpha, we mutated five putative Ca2+ ligands (D187N, D193N, D246N, D248N, and D254N) and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration of PKC-alpha. Altered properties of these mutants indicate that individual Ca2+ ions and their ligands have different roles in the membrane binding and activation of PKC-alpha. The binding of Ca2+ to Asp187, Asp193, and Asp246 of PKC-alpha is important for the initial binding of protein to membrane surfaces. On the other hand, the binding of another Ca2+ to Asp187, Asp246, Asp248, and Asp254 induces the conformational change of PKC-alpha, which in turn triggers its membrane penetration and activation. Among these Ca2+ ligands, Asp246 was shown to be most essential for both membrane binding and activation of PKC-alpha, presumably due to its coordination to multiple Ca2+ ions. Furthermore, to identify the residues in the C2 domain that are involved in membrane binding of PKC-alpha, we mutated four putative membrane binding residues (Trp245, Trp247, Arg249, and Arg252). Membrane binding and enzymatic properties of two double-site mutants (W245A/W247A and R249A/R252A) indicate that Arg249 and Arg252 are involved in electrostatic interactions of PKC-alpha with anionic membranes, whereas Trp245 and Trp247 participate in its penetration into membranes and resulting hydrophobic interactions. Taken together, these studies provide the first experimental evidence for the role of C2 domain of conventional PKC as a membrane docking unit as well as a module that triggers conformational changes to activate the protein.     

Transformation of mycosis fungoides: T-cell receptor beta gene analysis demonstrates a common clonal origin for plaque-type mycosis fungoides and CD30+ large-cell lymphoma

We investigated the consequences of Sr2+ binding to the transport sites of sarcoplasmic reticulum (SR) Ca(2+)-ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled with fluorescein 5'-isothiocyanate (FITC), we found that the Sr(2+)-induced a drop in the fluorescein fluorescence of this FITC-labeled ATPase shifted toward lower Sr2+ concentrations than the Sr(2+)-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describing the Sr(2+)-dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr2+ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr2+ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr(2+)-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr2+ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.     

Reduced positive feedback regulation between myosin crossbridge and cardiac troponin C in fast skeletal myofibrils

Several studies have shown that substitution of cardiac troponin C into fast skeletal muscle causes a marked reduction in cooperativity of Ca(2+)-activation of both myofibrillar ATPase and tension development. To clarify the underlying mechanisms, in the present study, Ca2+ binding to cardiac troponin C inserted into fast skeletal myofibrils was measured. Two classes of binding sites with different affinities (classes 1 and 2) were clearly identified, which were equivalent stoichiometrically to the two high-affinity sites (sites III and IV) and a single low-affinity site (site II) of troponin C, respectively. Ca2+ binding to class-2 sites and Ca(2+)-activation of myofibrillar ATPase occurred in roughly the same Ca2+ concentration range, indicating that site II is responsible for Ca2+ -regulation. Myosin crossbridge interactions with actin, both in the presence and absence of ATP, enhanced the Ca2+ binding affinity of only class-2 sites. These effects of myosin crossbridges, however, were much smaller than the effects on the Ca2+ binding to the low-affinity sites of fast skeletal troponin C, which are responsible for regulating fast skeletal myofibrillar ATPase. These findings provide strong evidence that the reduction in the cooperative response to Ca2+ upon substituting cardiac troponin C into fast skeletal myofibrils is due to a decrease in the positive feedback interaction between myosin crossbridge attachment and Ca2+ binding to the regulatory site of troponin C.     

Calcium ion binding to thrombospondin 1

We have quantified the binding of Ca2+ to platelet thrombospondin 1 (TSP1) using equilibrium dialysis with 45CaCl2. Ca2+ binding to TSP1 was found to be cooperative with 10% occupancy at 15-20 microM CaCl2, 90% occupancy at 100 microM CaCl2, and a Hill coefficient of 2.4 +/- 0.2 The average apparent Kd was 52 +/- 5 microM. Maximum binding, assuming Mr = 450,000 and epsilon = 0.918 (A280/mg/ml), was 35 +/- 3 Ca2+/TSP1. This value is close to the 33 sites (11 per subunit) predicted based on homology of the epidermal growth factor (1 site) and aspartate-rich (10 sites) regions to known Ca2+ binding sequences. Ca2+ protected the aspartate-rich region from trypsin proteolysis, but not until nearly all of the Ca2+ binding sites were filled. At lower occupancy of Ca2+ binding sites, several limited tryptic digest products were obtained. This finding and the previous demonstration of extensive thiol-disulfide isomerization within the aspartate-rich regions suggest that subregions of the aspartate-rich region are stabilized in different conformers. Zn2+, Cu2+, Mn2+, Mg2+, Co2+, Cd2+, and Ba2+ were tested for their ability to modulate Ca2+ binding and protease sensitivity of TSP1. Zn2+ inhibited 40% of the Ca2+ binding but neither protected TSP1 from trypsin proteolysis, nor labilized TSP1 toward trypsin proteolysis. These results provide direct evidence for high capacity, cooperative and specific binding of Ca2+ to conformationally labile aspartate-rich repeats of TSP1.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

Hydrophobic core substitutions in calbindin D9k: effects on Ca2+ binding and dissociation

Hydrophobic core residues have a marked influence on the Ca2+-binding properties of calbindin D9k, even though there are no direct contacts between these residues and the bound Ca2+ ions. Eleven different mutants with substitutions in the hydrophobic core were produced, and their equilibrium Ca2+-binding constants measured from Ca2+ titrations in the presence of chromophoric chelators. The Ca2+-dissociation rate constants were estimated from Ca2+ titrations followed by 1H NMR1 and were measured more accurately using stopped-flow fluorescence. The parameters were measured at four KCl concentrations to assess the salt dependence of the perturbations. The high similarity between the NMR spectra of mutants and wild-type calbindin D9k suggests that the structure is largely unperturbed by the substitutions. More detailed NMR investigations of the mutant in which Val61 is substituted by Ala showed that the mutation causes only very minimal perturbations in the immediate vicinity of residue 61. Substitutions of alanines or glycines for bulky residues in the center of the core were found to have significant effects on both Ca2+ affinity and dissociation rates. These substitutions caused a reduction in affinity and an increase in off-rate. Small effects, both increases and decreases, were observed for substitutions involving residues far from the Ca2+ sites and toward the outer part of the hydrophobic core. The mutant with the substitution Phe66 --> Trp behaved differently from all other mutants, and displayed a 25-fold increase in overall affinity of binding two Ca2+ ions and a 6-fold reduction in calcium dissociation rate. A strong correlation (R = 0.94) was found between the observed Ca2+-dissociation rates and affinities, as well as between the salt dependence of the off-rate and the distance to the nearest Ca2+-coordinating atom. There was also a strong correlation (R = 0.95) between the Ca2+ affinity and stability of the Ca2+ state and a correlation (R = 0. 69) between the Ca2+ affinity and stability of the apo state, as calculated from the results in the present and preceding paper in this issue [Julenius, K., Thulin, E., Linse, S., and Finn, B. E. (1998) Biochemistry 37, 8915-8925]. The change in salt dependencies of koff and cooperativity were most pronounced for residues completely buried in the core of the protein (solvent accessible surface area approximately 0). Altogether, the results suggest that the hydrophobic core residues promote Ca2+ binding both by contributing to the preformation of the Ca2+ sites in the apo state and by preferentially stabilizing the Ca2+-bound state.     

Cation binding and conformation of tryptic fragments of Nereis sarcoplasmic calcium-binding protein: calcium-induced homo- and heterodimerization

Nereis sarcoplasmic calcium-binding protein (NSCP) is a compact 20-kDa protein that competitively binds three Ca2+ or Mg2+ ions and displays strong positive cooperativity. Its three-dimensional structure is known. It thus constitutes a good model for the study of intramolecular information transduction. Here we probed its domain structure and interaction between domains using fragments obtained by controlled proteolysis. The metal-free form, but not the Ca2+ or Mg2+ form, is sensitive to trypsin proteolysis and is preferentially cleaved at two peptide bonds in the middle of the protein. The N-terminal fragment 1-80 (T1-80) and the C-terminal fragment 90-174 (T90-174) were purified to electrophoretic homogeneity. T1-80, which consists of a paired EF-hand domain, binds one Ca2+ with Ka = 3.1 x 10(5) M-1; entropy increase is the main driving force of complex formation. Circular dichroism indicates that T1-80 is rich in secondary structure, irrespective of the Ca2+ saturation. Ca2+ binding provokes a difference spectrum which is similar to that observed in the intact protein. These data suggest that this N-terminal domain constitutes the stable structural nucleus in NSCP to which the first Ca2+ binds. T90-174 binds two Ca2+ ions with Ka = 3.2 x 10(4) M-1; the enthalpy change contributes predominantly to the binding process. Metal-free T90-174 is mostly in random coil but converts to an alpha-helical-rich conformation upon Ca2+ binding. Ca2+ binding to T1-80 provokes a red-shift and intensity decrease of the Trp fluorescence but a blue-shift and intensity increase in T90-174.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of calcium-bound rat S100B(betabeta) as determined by nuclear magnetic resonance spectroscopy,

The three-dimensional structure of Ca2+-bound rat S100B(betabeta) has been determined using data from a series of two-dimensional (2D), three-dimensional (3D), and four-dimensional (4D) nuclear magnetic resonance (NMR) experiments. Each S100beta subunit (91 residues) contains four helixes (helix 1, E2-R20; helix 2, K29-N38; helix 3, Q50-D61; and helix 4, F70-A83) and one antiparallel beta-sheet (strand 1, K26-K28; and strand 2, E67-D69) which brings the normal and pseudo EF-hands together. As found previously for rat apo-S100B(betabeta) [Drohat, A. C., et al. (1996) Biochemistry 35, 11577-11588], helixes 1, 1', 4, and 4' associate to form an X-type four-helix bundle at the symmetric dimer interface. Additionally, Ca2+ binding does not significantly change the interhelical angle of helixes 1 and 2 in the pseudo EF-hand (apo, Omega1-2 = 132 +/- 4 degrees; and Ca2+-bound, Omega1-2 = 137 +/- 5 degrees). However, the interhelical angle of helixes 3 and 4 in the normal EF-hand (Omega3-4 = 106 +/- 4 degrees) changed significantly upon the addition of Ca2+ (DeltaOmega3-4 = 112 +/- 5 degrees) and is similar to that of the Ca2+-bound EF-hands in calbindin D9K, calmodulin, and troponin (84 degrees 相似文献   

Binding of Ca2+ and Zn2+ to human nuclear S100A2 and mutant proteins

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.     

Dynamics and thermodynamics of the regulatory domain of human cardiac troponin C in the apo- and calcium-saturated states

The contraction of cardiac and skeletal muscles is triggered by the binding of Ca2+ to their respective troponin C (TnC) proteins. Recent structural data of both cardiac and skeletal TnC in both the apo and Ca2+ states have revealed that the response to Ca2+ is fundamentally different for these two proteins. For skeletal TnC, binding of two Ca2+ to sites 1 and 2 leads to large changes in the structure, resulting in the exposure of a hydrophobic surface. For cardiac TnC, Ca2+ binds site 2 only, as site 1 is inactive, and the structures show that the Ca2+-induced changes are much smaller and do not result in the exposure of a large hydrophobic surface. To understand the differences between regulation of skeletal and cardiac muscle, we have investigated the effect of Ca2+ binding on the dynamics and thermodynamics of the regulatory N-domain of cardiac TnC (cNTnC) using backbone 15N nuclear magnetic resonance relaxation measurements for comparison to the skeletal system. Analysis of the relaxation data allows for the estimation of the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy of the Ca2+-binding sites on a per residue basis, which can be related to the structural features of the sites. The results indicate that binding of Ca2+ to the functional site in cNTnC makes the site more rigid with respect to high-frequency motions; this corresponds to a decrease in the conformational entropy (TdeltaS) of the site by 2.2 kcal mol(-1). Although site 1 is defunct, binding to site 2 also decreases the conformational entropy in the nonfunctional site by 0.5 kcal mol(-1). The results indicate that the Ca2+-binding sites in the regulatory domain are structurally and energetically coupled despite the inability of site 1 to bind Ca2+. Comparison between the cardiac and skeletal isoforms in the apo state shows that there is a decrease in conformational entropy of 0.9 kcal mol(-1) for site 1 of cNTnC and little difference for site 2.     

The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.     

Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1. 1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1. 1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens

Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obtained from structure energy minimization calculations based on known three-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme-substrate complex, in which the beta-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the beta-flank are established and the correct alignment on the alpha-flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

Spectroscopic studies of the interaction of Ca2+-ATPase-peptides with dodecyl maltoside and its brominated analog

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase comprises ten putative transmembrane spans (M1-M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding: peptide M6 (amino acid residues 785-810), peptide M7-L (amino acid residues 808-847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818-847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl beta-D-maltoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur J. Biochem. 241, 343-354]. Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7,8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solubilized Ca2+-ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short alpha-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.