Structural differences in the two calcium binding sites of the porcine intestinal calcium binding protein: a multinuclear NMR study

Cadmium-113 and calcium-43 NMR spectra of Cd2+ and Ca2+ bound to the porcine intestinal calcium binding protein (ICaBP; Mr 9000) contain two resonances. The first resonance is characterized by NMR parameters resembling those found for these cations bound to proteins containing the typical helix-loop-helix calcium binding domains of parvalbumin, calmodulin, and troponin C, which are defined as EF-hands by Kretsinger [Kretsinger, R. H. (1976) Annu. Rev. Biochem. 45, 239]. The second resonance in both spectra has a unique chemical shift and is consequently assigned to the metal ion bound in the N-terminal site of ICaBP. This site is characterized by an insertion of a proline in the loop of the helix-loop-helix domain and will be called the pseudo-EF-hand site. The binding of Cd2+ to the apo form of ICaBP is sequential. The EF-hand site is filled first. Both binding sites have similar, but not identical, affinities for Ca2+: at a Ca2+ to protein ratio of 1:1, 65% of the ion is bound in the EF-hand site and 35% in the pseudo-EF-hand site. The two sites do not appear to act independently; thus, replacement of Ca2+ or Cd2+ by La3+ in the EF-hand site causes changes in the environment of the ions in the pseudo-EF-hand site. In addition, the chemical shift of Cd2+ bound to the EF-hand site is dependent on the presence or absence of Ca2+ or Cd2+ in the pseudo-EF-hand site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal-ion binding to parvalbumin. A 113Cd-n.m.r. study of the binding of different lanthanide ions.

113Cd-n.m.r. studies were used to investigate the binding of the lanthanide ions La3+, Gd3+, Tb3+, Yb3+ and Lu3+ to parvalbumins. It was shown that lanthanide ions with a smaller ionic radius bind sequentially to Cd2+-saturated parvalbumin, whereas those with a larger ionic radius bind with similar affinity to both the CD site and the EF site. The smallest ion, Lu3+, does in fact not compete significantly with Cd2+ for the CD site in carp parvalbumin, but appears to bind only to the EF site. This preference of the smaller lanthanide ions for the EF site was used to assign the n.m.r. signals for protein-bound 113Cd. By using Cd n.m.r. and Tb3+ fluorescence it was also shown for alpha-lineage parvalbumin from pike that these proteins possess a third site that can bind lanthanide ions. This site is, however, much weaker than in the beta-lineage parvalbumins. It was used to assign the 113Cd resonances from protein-bound Cd2+ ions in the spectrum of pike pI5.0 parvalbumin.     

113Cd NMR study of bovine prothrombin fragment 1 and factor X

The interaction of Cd2+ with bovine prothrombin fragment 1, prothrombin intermediate 1, factor X, and a modified (Gla-domainless) factor X has been studied with 113Cd NMR. All the 113Cd resonances observed in this study were in the chemical shift range expected for oxygen ligands, suggesting that cadmium is binding at the same sites where calcium binds. Both fragment 1 and factor X displayed two major resonances, one near 10 ppm from 113Cd2+ that did not exchange rapidly with unbound 113Cd2+ (the high-affinity, or H, resonance) and one near -15 ppm from 113Cd2+ that exchanged rapidly with unbound 113Cd2+ (the low-affinity, or L, resonance). The difference between the chemical shift of the H resonance and the chemical shift range of -90 to -125 ppm that has been reported for three other small calcium-binding proteins is postulated to be due to different coordination geometries for monocarboxylate and dicarboxylate ligands; Cd2+ binds to fragment 1 and factor X through the dicarboxylate side chains of gamma-carboxyglutamate (Gla) residues. This allows contribution of only one oxygen per carboxyl group. At least one of the first few 113Cd2+ ions bound to fragment 1 did not appear in the 113Cd NMR spectrum until a total of five 113Cd2+ had been added. This could be due to exchange broadening of initial 113Cd2+ resonances due to sharing of ligands among several sites. Filling all sites would then restrict ligand exchange. Addition of Zn2+ displaced 113Cd2+ from the H resonance sites. Factor X did not display the interactions among ion binding sites proposed for fragment 1.     

Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted 113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. 113Cd resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of the resonances to the transition metal (S1) and calcium (S2) sites were unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and S2 sites, respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH fail to show a 113Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.     

113Cd nuclear magnetic resonance of Cd(II) alkaline phosphatases

113Cd NMR spectra of 113Cd(II)-substituted Escherichia coli alkaline phosphatase have been recorded over a range of pH values, levels of metal site occupancy, and states of phosphorylation. Under all conditions resonances attributable to cadmium specifically bound at one or more of the three pairs of metal-binding sites (A, B, and C sites) are detected. By following changes in both the 113Cd and 31P NMR spectra of 113Cd(II)2 alkaline phosphatase during and after phosphorylation, it has been possible to assign the cadmium resonance that occurs between 140 and 170 ppm to Cd(II) bound to the A or catalytic site of the enzyme and the resonance occurring between 51 and 76 ppm to Cd(II) bound to B site, which from x-ray data is located 3.9 A from the A site. The kinetics of phosphorylation show that cadmium migration from the A site of one subunit to the B site of the second subunit follows and is a consequence of phosphate binding, thus precluding the migration as a sufficient explanation for half-of-the-sites reactivity. Rather, there is evidence for subunit-subunit interaction rendering the phosphate binding sites inequivalent. Although one metal ion, at A site, is sufficient for phosphate binding and phosphorylation, the presence of a second metal ion at B site greatly enhances the rate of phosphorylation. In the absence of phosphate, occupation of the lower affinity B and C sites produces exchange broadening of the cadmium resonances. Phosphorylation abolishes this exchange modulation. Magnesium at high concentration broadens the resonances to the point of undetectability. The chemical shift of 113Cd(II) in both A and B sites (but not C site) is different depending on the state of the bound phosphate (whether covalently or noncovalently bound) and gives separate resonances for each form. Care must be taken in attributing the initial distribution of cadmium or phosphate in the reconstituted enzyme to that of the equilibrium species in samples reconstituted from apoenzyme. Both 113Cd NMR and 31P NMR show that some conformational changes consequent to metal ion or phosphate binding require several days before the final equilibrium species is formed.     

Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Preparation and 113Cd NMR studies of homogeneous reconstituted metallothionein: reaffirmation of the two-cluster arrangement of metals

113Cd NMR analysis of rabbit liver metallothionein 2 reconstituted with 113Cd at all seven binding sites has previously indicated that the metals are arranged in two metal-thiolate clusters [Otvos, J.D., & Armitage, I.M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. Spectra of the protein always contained more than seven resonances, however, suggesting the samples were in some way heterogeneous. Results of a recent study of 113Cd metallothionein reconstituted in a different manner but also giving spectra with more than seven resonances have been interpreted as arguing against the two-cluster model of metal binding and in favor of a model in which structural flexibility of the protein allows many configurational substates of the cluster(s) to coexist [Vasak, M., Hawkes, G.E., Nicholson, J.K., & Sadler, P.J. (1985) Biochemistry 24, 740-747]. Data are presented here that indicate that dimers and larger oligomers of metallothionein formed as byproducts of metal reconstitution are the likely source of at least some of the 113Cd resonances attributed by these workers to configurational substrates. Removal of the contaminating oligomers by gel filtration yields a verifiably homogeneous protein whose 113Cd spectrum consists of seven resonances of comparable intensity. Unambiguous confirmation of the existence and structures of the two previously proposed metal-thiolate clusters was obtained by two-dimensional chemical shift correlation spectroscopy and spectral simulation of the 113Cd-113Cd splitting patterns of the individual resonances.     

113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

Cadmium-substituted skeletal troponin C. Cadmium-113 NMR spectroscopy and metal binding investigations

The binding of cadmium to skeletal troponin C (STnC) has been measured by equilibrium binding and by 113Cd NMR spectroscopy. The equilibrium binding experiments have shown that there are two cadmium binding sites on STnC with a high affinity for Cd2+ (KCd congruent to 10(7) M-1) and two with a lower affinity for Cd2+ (KCd congruent to 10(3) M-1). The former binding constant is comparable to Ca2+ binding to the Ca2+-Mg2+ (structural) sites of STnC and the latter is about a factor of one hundred less than Ca2+ binding to the Ca2+-specific (regulatory) sites of STnC. In the presence of Mg2+ the affinity of Cd2+ for the higher affinity sites was lowered, yielding a KMg of approximately 10(3) M-1. These data clearly suggest that the two sites with high affinity for Cd2+ are the same as the Ca2+-Mg2+ sites (Zot, H., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The 113Cd NMR is shown to be temperature-dependent. The room temperature spectrum consists of two resonances at -107.8 and -112.7 ppm with respect to a 0.1 M solution of Cd(ClO4)2. Lowering the temperature to 4 degrees C alters the cadmium exchange dynamics, and results in a four line 113Cd spectrum. The two new resonances at -103.1 and -109.8 ppm probably arise from cadmium binding to the Ca2+-specific (regulatory) sites on STnC; whereas, the resonances at -107.8 and -112.7 ppm correspond to cadmium binding at the Ca2+-Mg2+ (structural) sites, respectively. When the 113Cd2+-substituted protein was titrated with Ca2+, the two resonances corresponding to the high affinity sites were reduced in intensity, followed by a reduction in intensity of the lower affinity Cd2+ sites. Based on the assignments made here and the known binding constants of STnC for Ca2+ (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633) and the Cd2+ affinities reported here, one would not predict these results. Ca2+ should have first bound to the sites with the lower affinity Cd2+. Since the direct binding experiments clearly demonstrate that the high affinity Cd2+ sites are the Ca2+-Mg2+ sites, we can only conclude that Cd2+ binding to the protein (probably to the lower affinity Ca2+-specific sites) dramatically alters the affinity of the Ca2+-Mg2+ sites for Ca2+. It is suggested that an allosteric coupling network exists between all classes of binding sites.     

Manganese (II) electron spin resonance and cadmium-113 nuclear magnetic resonance evidence for the nature of the calcium binding site in alpha-lactalbumins

Bovine and goat alpha-lactalbumins were substituted with 113Cd(II) or Mn(II) at the strong calcium site [Murakami, K., Andree, P.J., & Berliner, L.J. (1982) Biochemistry 21, 5488-5494] and studied by 113 Cd NMR and electron spin resonance. The 113Cd chemical shifts were in the -80 to -85 ppm range vs. Cd(ClO4)2, which was almost identical with that found for several nearly octahedral (oxygen-coordinated) calcium binding proteins such as calmodulin, parvalbumin, and troponin C. The electron spin resonance spectra of bound Mn(II)-alpha-lactalbumin complexes at 9 or 35 GHz were also confirmatory of a highly symmetric (cubic) environment around the Mn(II) with only slight distortions. The near identity of this site in alpha-lactalbumin to those of calcium binding proteins containing an "EF hand domain" was remarkable despite the absence of such a domain sequence in the alpha-lactalbumin structure.     

Oncomodulin. 1H NMR and optical stopped-flow spectroscopic studies of its solution conformation and metal-binding properties

As deduced from its 1H NMR spectrum, oncomodulin's solution conformation is very similar to the tertiary structure of other single domain 2-site calcium-binding proteins of the troponin C class. Despite its extensive amino acid sequence homology with parvalbumins, however, oncomodulin differs significantly from these proteins in its Ca(II)----Ln(III) exchange characteristics. Although the relative affinity of Lu(III) for the EF site of Ca2-oncomodulin was normal, beta Lu:EF/beta Ca:EF being 175 +/- 15, displacement of Ca(II) from the CD site was not favored, beta Lu:CD/beta Ca:CD being 1.2 +/- 0.1. Lineshape analyses of several 1H NMR resonances generated by the Lu(III) titration of Ca2-oncomodulin indicated that Ca(II)----Ln(III) exchange at the CD site was 15-20 s-1, approximately 100 times faster than exchange at the CD site of parvalbumins. Analyses of the distribution of metal-bound oncomodulin species showed that Ca(II)----Lu(III) exchange was cooperative, the coefficient of cooperativity being estimated as 5 +/- 1. The kinetics of the release of Yb(III) from oncomodulin as measured by optical stopped-flow techniques corroborated the observed cooperativity in metal binding; the off-rate constant of Yb(III) from the EF site of Yb2-oncomodulin was 0.0036 s-1, approximately 19 times slower than the release of Yb(III) from the EF site of Ca1Yb1-oncomodulin. We attribute part of the reduced preference of small Ln(III)s for the CD site of oncomodulin to a combination of this site's inherent incompressibility (Williams, T.C., Corson, D.C. & Sykes, B.D. (1984) J. Am. Chem. Soc. 106, 5698-5702) and the Glu----Asp substitution at sequence position 59, the residue which chelates metal at the -X coordination position. Like the CD site in oncomodulin, site III in troponin C has not only a lower affinity for calcium relative to the CD site of parvalbumins but also aspartic acid at its -X position; a water molecule bridges the gap between bound metal and the carboxyl group of the relatively short side chain of Asp-114 (Herzberg, O. & James, M. N. G. (1985) Biochemistry 24, 5298-5302). Hence, we suggest that Asp-59 in oncomodulin binds metal only indirectly through an intervening water molecule, a proposal which is consistent with the CD site's reduced affinity for ions the size of Ca(II) or smaller.     

1H, 113Cd, and 31P NMR of osteocalcin (bovine gamma-carboxyglutamic acid containing protein)

The 1H (500-MHz), 113Cd (44-MHz), and 31P (81-MHz) NMR spectra of the bovine gamma-carboxyglutamate- (Gla-) containing protein osteocalcin and its Ca(II) and Cd(II) complexes in solution have been obtained. The 1H NMR spectrum of the native protein shows narrow resonances and a highly resolved multiplet structure suggesting rotational freedom of the side chains. In comparison to the simulated 1H NMR spectrum of a random polypeptide chain of the same amino acid composition, there is moderate chemical shift dispersion, indicating some conformational restraints to be present. Ca(II) binding broadens all 1H resonances, so severely at four Ca(II) ions per molecule that few structural conclusions can be made. Cd(II) substituted for Ca(II) has the same effect, and 113Cd NMR shows the Cd(II) to be in intermediate chemical exchange on the chemical shift time scale. Estimates of the chemical exchange rates required for 1H and 113Cd line broadening suggest a range of Kd values for the metal ion complexes from 10(-6) M to as high as 10(-3) M depending on the number of metal ions bound. Alternatively, 1H line broadening could be explained by relatively slow conformational fluxes in the protein induced by labile metal ion binding to one or more sites. Cd(II) when used to form a cadmium-phosphate mineral analogous to hydroxylapatite results in a crystal lattice that removes osteocalcin from solution just as effectively as hydroxylapatite. 113Cd(II) exchange at the binding sites of osteocalcin in solution is slowed dramatically by the addition of HPO4(2-). 31P NMR shows the interaction of phosphate with the protein to require the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.     

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.     

Characterization of the zinc binding site of bacterial phosphotriesterase.

The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.     

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.     

The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding

Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics. We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase. The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra. Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals. For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions. The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry. The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed. The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme.     

Comparative Cd-113 nuclear magnetic resonance studies of Cd(II)-substituted blue copper proteins

The 113Cd NMR spectra of plastocyanin (Spinacea), stellacyanin (Rhus vernicifera), and two azurins (Pseudomonas aeruginosa and Alcaligenes faecalis) have been measured after introducing Cd(II) into the blue copper-binding sites. Relative to Cd(C1O4)2 the chemical shifts are 432, 380, 372, and 379 ppm, respectively, all of which are found to be reasonable values for binding sites containing a cysteine thiolate ligand. The 113Cd resonances of the cadmium derivatives of stellacyanin and the azurins are so near the same that the proteins must present very similar metal-binding sites. In contrast the plastocyanin derivative resonates about 50 ppm further downfield which may signal a change in coordination number. The spin lattice relaxation times of the 113Cd resonances are of the order of 0.1 s, and a major portion of the relaxation apparently occurs through the chemical shift anisotropy mechanism. At 13 degrees C the 113Cd resonance of Psuedomonas azurin shifts slightly downfield with increasing pH. This is explained by a small change in the environment about cadmium which occurs as a result of the conformational change that attends the titration of His-35.