Binding ability of a HHP-tagged protein towards Ni2+ studied by paramagnetic NMR relaxation: The possibility of obtaining long-range structure information

The binding ability of a protein with a metal binding tag towards Ni(2+) was investigated by longitudinal paramagnetic NMR relaxation, and the possibility of obtaining long-range structure information from the paramagnetic relaxation was explored. A protein with a well-defined solution structure (Escherichia coli thioredoxin) was used as the model system, and the peptide His-His-Pro (HHP) fused to the N-terminus of the protein was used as the metal binding tag. It was found that the tag forms a stable dimer complex with the paramagnetic Ni(2+) ion, where each metal ion binds two HHP-tagged protein molecules. However, it was also found that additional sites in the protein compete with the HHP-tag for the binding of the metal ion. These binding sites were identified as the side chain carboxylate groups of the aspartic and glutamic acid residues. Yet, the carboxylate groups bind the Ni(2+) ions considerably weaker than the HHP-tag, and only protons spatially close to the carboxylate sites are affected by the Ni(2+) ions bound to these groups. As for the protons that are unaffected by the carboxylate-bound Ni(2+) ions, it was found that the long-range distances derived from the paramagnetic relaxation enhancements are in good agreement with the solution structure of thioredoxin. Specifically, the obtained long-range paramagnetic distance constraints revealed that the dimer complex is asymmetric with different orientations of the two protein molecules relative to the Ni(2+) ion.     

Metal-protein interactions: structure information from Ni(2+)-induced pseudocontact shifts in a native nonmetalloprotein

Information about the structure of a native nonmetalloprotein was obtained from the pseudocontact shifts induced by a paramagnetic metal ion bound to the protein. The approach exploits the presence of metal binding sites on the surface of the protein. Using Escherichia coli thioredoxin as a model protein, we show that potential binding sites can be identified using the Cu(2+) ion, and that pseudocontact shifts induced by a Ni(2+) ion bound to one of these sites can provide valuable long-range structure information about the protein.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献   

Characterization of calcium binding properties of lithostathine

The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with (45)Ca(2+) showed that LIT binds Ca(2+) with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO(2+)) ion as a paramagnetic substitute for Ca(2+) also showed that VO(2+) binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO(2+) competes with Ca(2+) in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95-100%) of binding of Ca(2+) and VO(2+), showing that these residues are critical for calcium binding by LIT.     

Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.     

Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase

Calcineurin, a calmodulin-regulated phosphatase, is composed of two distinct subunits (A and B) and requires certain metal ions for activity. The binding of the two most potent activators, Ni2+ and Mn2+, to calcineurin and its subunits has been studied. Incubation of the protein with 63Ni2+ (or 54Mn2+) followed by gel filtration to separate free and protein-bound ions indicated that calcineurin could maximally bind 2 mol/mol of Ni2+ or Mn2+. While isolated A subunit also bound 2 mol/mol of Ni2+, no Mn2+ binding was demonstrated for either isolated A or B subunit. When bindings were monitored by nitrocellulose filter assay, only 1 mol/mol bound Ni2+ or Mn2+ was detected, suggesting that the two Ni2+ (or Mn2+) binding sites had different relative affinities and that only metal ions bound at the higher affinity sites were detected by the filter assay. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the filter assay-measured Ni2+ (or Mn2+) binding by only 30%. Preincubation of the protein with Zn2+ decreased the filter assay-measured Ni2+ or Mn2+ binding by 90 or 17%, respectively. The results suggest that the higher affinity sites are a Ni2+-specific site and a distinct Mn2+-specific site. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the gel filtration-determined Ni2+ (or Mn2+) binding from 2 to 1 mol/mol suggesting that calcineurin also contains a site which binds either metal ion. The time course of Ni2+ (or Mn2+) binding was correlated with that of the enzyme activation, and the extent of deactivation of the Ni2+-activated calcineurin by EDTA or by incubation with Ca2+ and calmodulin (Pallen, C. J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141) was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation.     

Escherichia coli thioredoxin inhibition by cadmium: two mutually exclusive binding sites involving Cys32 and Asp26.

Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd(2+) led us to investigate the thioredoxin-cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 x 10(6) m(-1) and 1 x 10(6) m(-1). For both sites, a proton was released upon Cd(2+) binding. One mole of Cd(2+) per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd(2+) binding at either site totally inhibited the thiol-disulfide transferase activity of Trx. The absence of Cd(2+) interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd(2+) supported the role of Cys32 at the first site. The fluorescence profile of Cd(2+)-bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd(2+) was not coordinated with Cys32 and Cys35. From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pK(a) for a carboxylate (7.5/9.2). The pK(a) of the two residues Cys32 and Asp26 have been shown to be interdependent [Chivers, T. P. (1997) Biochemistry36, 14985-14991]. A mechanism is proposed in which Cd(2+) binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pK(a) of Asp26 and its deprotonation. Conversely, interaction between the carboxylate group of Asp26 and Cd(2+) at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd(2+) inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26.     

Simultaneous multiple element detection by particle beam/hollow cathode-optical emission spectroscopy as a tool for metallomic studies: determinations of metal binding with apo-transferrin

Particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) is presented as a tool for the determination of metal ion loading in transferrin (Tf). The elemental specificity of optical emission spectroscopy provides a means of assessing metal ion concentrations as well as the relative amounts of metal per unit protein concentration (up to 2 moles of Fe per mole of protein). The PB/HC-OES method allows for the simultaneous detection of metal content (Fe (I) 371.99, Ni (I) 341.41 nm, Zn (I) 213.86 nm, and Ag (I) 338.28 nm in this case), as well as elemental carbon and sulfur (C (I) 156.14 nm and S (I) 180.73 nm) that are reflective of the protein composition and concentration. Quantification for the metal species is based on calibration functions derived from aqueous solutions, with limits of detection for the entire suite being less than 1.0 μM. Determinations in this manner eliminate much of the ambiguity inherent in UV-VIS absorbance determinations of Tf metal binding. Validation of this method is obtained by analyzing loading response of Fe(3+) into Tf using the PB/HC-OES method and comparing the results with those of the standard UV-VIS absorbance method. Maximum Fe(3+) loading of Tf (based on the number of available binding sites) was determined to be 71.2 ± 4.7% by the PB/HC-OES method and 67.5 ± 2.5% for the UV-VIS absorbance method. Element emission ratios between the dopant metals and the carbon and sulfur protein constituents allow for concentration independent determinations of metal binding into Tf. Loading percentages were determined for Ni(2+), Zn(2+), and Ag(+) into Tf with maximum loading values of 19.5 ± 0.4%, 41.0 ± 4.4%, and 141.2 ± 4.3%, respectively. While of no apparent biological significance, Ag(+) presents an interesting case as a surrogate for Pt(2+), whose binding with Tf has shown to be quite different from the other metals. A different mode from the others is indeed observed, and is consistent with conjecture on the Pt(2+) mechanisms. Competitive binding studies not easily performed using absorbance spectroscopy are easily performed by simultaneous, multielement analysis, reflective of the metals and the protein content. In this work, there is clear competition between and Fe(3+) and Zn(2+) for binding in the C-terminus lobe of Tf, while Ni(2+) binds within the N-terminus lobe. Addition of Ag(+) to this mixture does not affect the other metals' distributions, but reflects binding at other protein sites.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

Functional studies of the Mycobacterium tuberculosis iron-dependent regulator

The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria. IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M. tuberculosis virulence. In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays. The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated. Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly. Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+). Binding of the second metal ion fully activates IdeR for binding to the fxbA operator. The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+). Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites. These results provide insight into the possible roles for each metal binding site in IdeR activation.     

Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme.

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) Biochemistry 35, 4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 17O-labeled forms of phosphoenolpyruvate show that Mn(2+), bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of 2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of 2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn(2+) gives lower maximal activity than Mg(2+), apparently due to slow release of Zn(2+) from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn(2+) from the enzyme.     

The protein gp74 from the bacteriophage HK97 functions as a HNH endonuclease

The last gene in the genome of the bacteriophage HK97 encodes the protein gp74. We present data in this article that demonstrates, for the first time, that gp74 possesses HNH endonuclease activity. HNH endonucleases are small DNA binding and digestion proteins characterized by two His residues and an Asn residue. We demonstrate that gp74 cleaves lambda phage DNA at multiple sites and that gp74 requires divalent metals for its endonuclease activity. We also present intrinsic tryptophan fluorescence data that show direct binding of Ni(2+) to gp74. The activity of gp74 in the presence of Ni(2+) is significantly decreased below neutral pH, suggesting the presence of one or more His residues in metal binding and/or DNA digestion. Surprisingly, this pH-dependence of activity is not seen with Zn(2+) , suggesting a different mode of binding of Zn(2+) and Ni(2+) . This difference in activity may result from binding of a second Zn(2+) ion by a putative zinc finger in gp74 in addition to binding of a Zn(2+) ion by the HNH motif. These studies define the biochemical function of gp74 as an HNH endonuclease and provide a platform for determining the role of gp74 in life cycle of the bacteriophage HK97.     

Discovery of native metal ion sites located on the ferredoxin docking side of photosystem I

Photosystem I (PSI) is a large membrane protein that catalyzes light-driven electron transfer across the thylakoid membrane from plastocyanin located in the lumen to ferredoxin in the stroma. Metal analysis reveals that PSI isolated from the cyanobacterial membranes of Synechococcus leopoliensishas a near-stoichiometric 1 molar equiv of Zn (2+) per PSI monomer and two additional surface metal ion sites that favor Cu (2+) binding. Two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy reveals coupling to the so-called remote nitrogen of a single histidine coordinated to one of the Cu (2+) centers. EPR and X-ray absorption fine structure (XAFS) studies of 2Cu-PSI complexes reveal the direct interaction of ferredoxin with the Cu (2+) centers on PSI, establishing the location of native metal sites on the ferredoxin docking side of PSI. On the basis of these spectroscopic results and previously reported site-directed mutagenesis studies, inspection of the PSI crystal structure reveals a cluster of three highly conserved residues, His(D95), Glu(D103), and Asp(C23), as a likely Cu (2+) binding site. The discovery of surface metal sites on the acceptor side of PSI provides a unique opportunity to probe the stromal region of PSI and the interactions of PSI with its reaction partner, the soluble electron carrier protein ferredoxin.     

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.     

The nickel site of Bacillus pasteurii UreE, a urease metallo-chaperone, as revealed by metal-binding studies and X-ray absorption spectroscopy

UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

A conserved cis-proline precludes metal binding by the active site thiolates in members of the thioredoxin family of proteins

Many thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.     

A paramagnetic probe to localize residues next to carboxylates on protein surfaces

It is shown that the paramagnetic properties of lanthanides can be exploited to obtain information on specific parts of a protein surface. Owing to the high affinity of coordinatively unsaturated lanthanide complexes for oxygen donors, carboxylate groups can be used as preferential targets for the interaction. The DO3A ligand is particularly useful in these studies, as it coordinates lanthanides in a heptadentate fashion, leaving two sites available for exogenous donors. A solution of a (15)N-labeled sample protein, calbindin D(9k) (75 residues), was titrated with up to 200% of Gd(III)-DO3A complex, and an inversion recovery (15)N-(1)H HSQC experiment was used to measure the paramagnetic contributions to the longitudinal relaxation rates of the amide protons. Relaxation data were used as distance constraints to estimate the number of interacting complexes and the occupancies of their binding sites. Four preferential interaction sites on the protein surface are found. Inspection of the various carboxylate side chains on the surface of the protein indicates that Gd(III)-DO3A interacts preferentially with carboxylate-rich regions, rather than with isolated carboxylates, suggesting the possibility of chelation of one Gd(III)-DO3A molecule by two carboxylate groups. Gd(III)-DO3A is thus a valuable semi-selective probe for clusters of negative charges on the protein surface.