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.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.     

113Cd nmr study of the metal cluster structure of human liver metallothionein

Cadmium-113 nuclear magnetic resonance (¹¹³Cd nmr) was used to elucidate the structural properties of the cadmium binding sites in human liver metallothionein. The isotopically labeled ¹¹³Cd-metallothionein was prepared by the in vitro exchange of the native metals (greater than 94% zinc) for ¹¹³CdCl₂ during isolation. The two isoproteins, MT-1 and MT-2, showed ¹¹³Cd nmr resonances in the chemical shift range 610–670 ppm. The multiplet structure of the resonances is due to two bond scalar interactions between adjacent ¹¹³Cd ions linked by cysteine thiolate ligands. Homonuclear ¹¹³Cd decoupling experiments allowed the determination of the metal cluster structure, which, similar to the rabbit liver metallothionein, consists of a four- and a three-metal cluster designated cluster A and cluster B, respectively. Chemical shift similarities in the ¹¹³Cd nmr spectra of the human, rabbit and calf liver MT-1 and MT-2 are observed, especially for cluster A. Small variations in chemical shifts are explained in terms of differences in the primary structure between the two human isoproteins.     

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.     

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.     

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.     

13C and 113Cd NMR studies of the chelation of metal ions by the calcium binding protein parvalbumin

13C NMR spectra are presented for the calcium binding protein parvalbumin (pI 4.25) from carp muscle in several different metal bound forms: with Ca2+ in both the CD and EF calcium binding sites, with Cd2+ in both sites, with 113Cd2+ in both sites, and with 113Cd2+ in the CD site and Lu3+ in the EF site. The different metals differentially shift the 13C NMR resonances of the protein ligands involved in chelation of the metal ion. In addition, direct 13C-113Cd spin-spin coupling is observed which allows the assignment of protein carbonyl and carboxyl 13C NMR resonances to ligands directly interacting with the metal ions in the CD and EF binding sites. The displacement of 113Cd2+ from the EF site by Lu3+ further allows these resonances to be assigned to the CD or EF site. The occupancy of the two sites in the two cadmium species and in the mixed Cd2+/Lu3+ species is verified by 113Cd NMR. The resolution in these 113Cd NMR spectra is sufficient to demonstrate direct interaction between the two metal binding sites.     

Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase

Metal binding at the activating site of rabbit muscle phosphoglucomutase has been studied by 31P, 7Li, and 113Cd NMR spectroscopy. A 7Li NMR signal of the binary Li+ complex of the phosphoenzyme was not observed probably because of rapid transverse relaxation of the bound ion due to chemical exchange with free Li+. The phosphoenzyme-Li+-glucose 6-phosphate ternary complex is more stable, kinetically, and yields a well-resolved peak from bound Li+ at -0.24 ppm from LiCl with a line width of 5 Hz and a T1 relaxation time of 0.51 +/- 0.07 s at 78 MHz. When glucose 1-phosphate was bound, instead, the chemical shift of bound 7Li+ was -0.13 ppm; and in the Li+ complex of the dephosphoenzyme and glucose bisphosphate a partially broadened 7Li+ peak appeared at -0.08 ppm. Thus, the bound metal ion has a somewhat different environment in each of these three ternary complexes. The 113Cd NMR signal of the binary Cd2+ complex of the phosphoenzyme appears at 22 ppm relative to Cd(ClO4)2 with a line width of 20 Hz at 44.4 MHz. Binding of substrate and formation of the Cd2+ complex of the dephosphoenzyme and glucose bisphosphate broaden the 113Cd NMR signal to 70 Hz and shift it to 75 ppm. The 53 ppm downfield shift upon the addition of substrate along with 1H NMR data suggests that one oxygen ligand to Cd2+ in the binary complex is replaced by a nitrogen ligand at some intermediate point in the enzymic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Thermolysin-inhibitor complexes examined by 31P and 113Cd NMR spectroscopy

Complexes between phosphoramidon (N-(alpha-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptoph an) and zinc thermolysin and between phosphoramidon or N-phosphoryl-L-leucineamide and 113Cd-substituted thermolysin have been examined by 31P and 113Cd NMR spectroscopy. 113Cd resonances are observed at 168 and 152 ppm for the phosphoramidon and N-phosphoryl-L-leucineamide complexes, respectively. There are large but different chemical shift anisotropy contributions to the 113Cd line widths for the two complexes, which reflect the known structural differences for the zinc-enzyme complexes. 113Cd-31P spin-spin coupling is also seen and differs for the two cadmium complexes, being larger, 28 Hz, for the bidentate N-phosphoryl-L-leucineamide ligand than for the monodentate phosphoramidon, 16 Hz. Large changes in chemical shift, 7.5-10.9 ppm, are seen for the 31P resonances of the inhibitors upon binding to the enzyme reflecting direct phosphoryl-metal ligation. Chemical shift anisotropy is the dominant relaxation mechanism for the 31P nuclei at 9.4 T, while the dipole-dipole contribution seems to be unaffected by a change of solvent from H2O to D2O.     

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)     

The metal binding site of zoocin A

Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site.On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2 +/- 0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.     

Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor

The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexpressed and purified from Escherichia coli previously. This fragment has been shown to contain 2.3 +/- 0.2 mol of Zn(II) per mole of protein [Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., & Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR(440-525)] with 113Cd(II) substituted for Zn(II) identifies 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus. The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands. Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear. Addition of EDTA to the protein restores the original two signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of cysteine ligands in metalloproteins using optical and NMR spectroscopy: cadmium-substituted rubredoxin as a model [Cd(CysS)4]2- center.

Optical and NMR methods are presented for the identification of cysteine ligands in Cd-substituted metalloproteins, in particular those containing zinc-fingerlike motifs, using Cd-substituted Desulfovibrio gigas rubredoxin (Cd-Rd) as a model [Cd(CysS)4]2- complex. The 113Cd NMR spectrum of Cd-Rd contains a single 113Cd resonance with a chemical shift position (723.6 ppm) consistent with tetrathiolate metal coordination. The proton chemical shifts of the four cysteine ligands were obtained from one-dimensional heteronuclear (1H-113Cd) multiple quantum coherence (HMQC) and total coherence spectroscopy (TOCSY)-relayed HMQC experiments. In addition, sequential assignments were made for two short cysteine-containing stretches of the polypeptide chain using a combination of homonuclear proton correlated spectroscopy, TOCSY, and nuclear Overhauser effect spectroscopy experiments, enabling sequence-specific heteronuclear 3J(1H beta-113Cd) coupling constants for each cysteine to be determined. The magnitude of these couplings (0-38 Hz) follows a Karplus-like dependence with respect to the H beta-C beta-S gamma-Cd dihedral angles, inferred from the crystal structure of the native protein. The difference absorption envelope (Cd-Rd vs. apo-Rd) reveals three distinct transitions with Gaussian-resolved maxima located at 213, 229, and 245 nm, which are paralleled by dichroic features in the corresponding difference CD and magnetic CD spectra. Based on the optical electronegativity theory of Jørgensen, the lowest energy transition has been attributed to a CysS-Cd(II) charge-transfer excitation (epsilon 245, 26,000 M-1 cm-1) with a molar extinction coefficient per cysteine of 6,500 M-1 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the form of Cd in the leaves of a superior Cd-accumulating ecotype of Thlaspi caerulescens using 113Cd-NMR

Thlaspi caerulescens (Ganges ecotype) is a known Cd hyperaccumulator, however, the ligands which coordinate to Cd ions in the leaves have not been identified. In the present study, the chemical form of Cd was investigated by using ¹¹³Cd-nuclear magnetic resonance (NMR) spectroscopy. Plants were grown hydroponically with a highly enriched ¹¹³Cd stable isotope. Measurements of ¹¹³Cd-NMR with intact leaves showed a signal at the chemical shift of around –16 ppm. Crude leaf sap also gave a similar chemical shift. Purification by gel filtration (Sephadex G-10), followed by cationic and anionic exchange chromatography, showed that Cd occurred only in the anionic fraction, which gave the same chemical shift as intact leaves. Further purification of the anionic fraction, combined with ¹¹³Cd- and ¹H-NMR studies, revealed that only the fraction containing malate showed a chemical shift similar to the intact leaves. These results indicate that Cd was coordinated mainly with malate in the leaves of T. caerulescens. The malate concentration in the leaves was not affected by increasing Cd concentration in the solution, suggesting that malate synthesis is not induced by Cd. Because the Cd-malate complex is relatively weak, we suggest that the complex forms inside the vacuoles as a result of an efficient tonoplast transport of Cd and a constitutively high concentration of malate in the vacuoles, and that the formation of the Cd-malate complex may lead to a decrease of subsequent Cd efflux to the cytoplasm.     

15N-labeled 5S RNA. Identification of uridine base pairs in Escherichia coli 5S RNA by 1H-15N multiple quantum NMR

Escherichia coli 5S RNA labeled with 15N at N3 of the uridines was isolated from the S phi-187 uracil auxotroph grown on a minimal medium supplemented with [3-15N]uracil. 1H-15N multiple quantum filtered and 2D chemical shift correlated spectra gave resonances for the uridine imino 1H-15N units whose protons were exchanging slowly with solvent. Peaks with 1H/15N shifts at 11.6/154.8, 11.7/155.0, 11.8/155.5, 12.1/155.0, and 12.2/155.0 ppm were assigned to GU interactions. Two labile high-field AU resonances at 12.6/156.8 and 12.8/157.3 ppm typical of AU pairs in a shielded environment at the end of a helix were seen. Intense AU signals were also found at 13.4/158.5 and 13.6/159.2 ppm where 1H-15N units in normal Watson-Crick pairs resonate. 1H resonances at 10.6 and 13.8 ppm were too weak, presumably because of exchange with water, to give peaks in chemical shift correlated spectra. 1H chemical shifts suggest that the resonance at 13.8 ppm represents a labile AU pair, while the resonance at 10.6 ppm is typical of a tertiary interaction between U and a tightly bound water or a phosphate residue. The NMR data are consistent with proposed secondary structures for 5S RNA.     

Evidence for a dynamic structure of human neuronal growth inhibitory factor and for major rearrangements of its metal-thiolate clusters.

Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.     

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.     

13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.     

Calcium binding to the isolated beta-hydroxyaspartic acid-containing epidermal growth factor-like domain of bovine factor X

Coagulation factor X is a vitamin K-dependent protein composed of discrete domains or modules. A proteolytically modified derivative of factor X that lacks the NH2-terminal gamma-carboxyglutamic acid (Gla)-containing region retains one Ca2+ binding site. To localize this Gla-independent Ca2+ binding site and to facilitate future studies aimed at elucidating structure-function relationship in the factor X molecule, we have devised a method to isolate the first beta-hydroxyaspartic acid (Hya)-containing epidermal growth factor (EGF)-like domain from proteolytic digests of bovine factor X performed under strictly controlled conditions. The EGF-like domain, corresponding to residues 45-86 in bovine factor X, was obtained in more than 50% recovery, and was at least 98% homogeneous as judged by NH2-terminal sequence analysis. Ca2+ binding to the isolated EGF-like domain was studied by 1H NMR spectroscopy. On binding of Ca2+ to the domain the resonances from Tyr-68 centered at 6.8 ppm were affected. The Ca2+ concentration dependence of the chemical shift was used to calculate the Ca2+ binding constant, resulting in a K alpha of 4 X 10(3) M-1 at pH 8.5 and 1 X 10(3) M-1 at pH 7.4, the higher value presumably reflecting an increase in negative surface charge due to deprotonation of a histidine residue with a pK alpha of 7.4. The NMR spectra gave no evidence of a conformational change in the EGF-like domain between pH 6 and 8.5.