Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT.     

Crystal structure of D-aminoacylase from Alcaligenes faecalis DA1. A novel subset of amidohydrolases and insights into the enzyme mechanism

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small beta-barrel, and a catalytic (betaalpha)(8)-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha/beta-barrel amidohydrolase superfamily, in which the beta-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys(96), His(220), and His(250), while the other is loosely chelated by His(67), His(69), and Cys(96). This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.     

Ions binding to S100 proteins. II. Conformational studies and calcium-induced conformational changes in S100 alpha alpha protein: the effect of acidic pH and calcium incubation on subunit exchange in S100a (alpha beta) protein

A rapid separation method for bovine brain S100 alpha alpha, S100a, and S100b protein using fast protein liquid chromatography on a Mono Q column and its application in preparation of a large amount of S100 alpha alpha protein are described. The conformation of S100 alpha alpha in the metal-free forms as well as in the presence of calcium were studied by UV absorption, circular dichroism, intrinsic fluorescence, sulfhydryl reactivity, and interaction with a hydrophobic fluorescent probe. The alpha-subunit appears to have nearly identical conformation in S100 alpha alpha and S100a protein dimers. We also confirmed that only the alpha-subunit exposes hydrophobic domains to solvent in the presence of calcium and that cysteine residues exposed upon Ca2+ binding to S100 proteins correspond to Cys 85 alpha and Cys 84 beta. Incubation of S100a with calcium and KCl proved that calcium binding to the putative calcium-binding sites (site I alpha, I beta) triggers a time- and temperature-dependent conformational change in the protein structure which decreases the antagonistic effect of KCl on calcium binding to sites II alpha and II beta and provokes subunit exchanges between protein dimers and the emergence of S100 alpha alpha and S100b (beta beta) proteins. Dynamic fluorescence measurements showed that incubating calcium at high S100a protein concentrations (greater than 10(-5) M) induces an apparent slow dimer-monomer equilibrium which might result in total subunit dissociation at lower protein concentrations. The effect of acidic pH on subunit dissociation in S100a protein (Morero, R. D., and Weber, G. (1982) Biochim. Biophys. Acta 703, 231-240) arises from conformational changes in the protein structure that are similar to those induced by Ca2+ incubation.     

Substrate-assisted cysteine deprotonation in the mechanism of dimethylargininase (DDAH) from Pseudomonas aeruginosa

The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N(omega)-methyl-l-arginine and N(omega),N(omega)-dimethyl-l-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK(a) of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK(a) values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k(cat) values indicates a much lower apparent pK(a) value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.     

Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity

ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved 392CPC394 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.     

pH-dependent structural change of reduced spinach plastocyanin studied by perturbed angular correlation of γ-rays and dynamic light scattering

In this study the pH-dependent structural changes of reduced spinach plastocyanin were investigated using perturbed angular correlation (PAC) of γ-rays and dynamic light scattering (DLS). PAC data of Ag-substituted plastocyanin indicated that the coordinating ligands are two histidine residues (His37, His87) and a cysteine residue (Cys84) in a planar configuration, whereas the methionine (Met92) found perpendicular to this plane is not a coordinating ligand at neutral pH. Two slightly different conformations with differences in the Cys–metal ion–His angles could be observed with PAC spectroscopy. At pH 5.3 a third coordination geometry appears which can be explained as the absence of the His87 residue and the coordination of Met92 as a ligand. With DLS the aggregation of reduced plastocyanin could be observed below pH 5.3, indicating that not only the metal binding site but also the aggregation properties of the protein change upon pH reduction. Both the structural changes at the metal binding site and the aggregation are shown to be reversible. These results support the hypothesis that the pH of the thylakoid lumen has to remain moderate during steady-state photosynthesis and indicate that low pH induced aggregation of plastocyanin might serve as a regulatory switch for photosynthesis.     

Prediction of structures of zinc‐binding proteins through explicit modeling of metal coordination geometry

Metal ions play an essential role in stabilizing protein structures and contributing to protein function. Ions such as zinc have well‐defined coordination geometries, but it has not been easy to take advantage of this knowledge in protein structure prediction efforts. Here, we present a computational method to predict structures of zinc‐binding proteins given knowledge of the positions of zinc‐coordinating residues in the amino acid sequence. The method takes advantage of the “atom‐tree” representation of molecular systems and modular architecture of the Rosetta3 software suite to incorporate explicit metal ion coordination geometry into previously developed de novo prediction and loop modeling protocols. Zinc cofactors are tethered to their interacting residues based on coordination geometries observed in natural zinc‐binding proteins. The incorporation of explicit zinc atoms and their coordination geometry in both de novo structure prediction and loop modeling significantly improves sampling near the native conformation. The method can be readily extended to predict protein structures bound to other metal and/or small chemical cofactors with well‐defined coordination or ligation geometry.     

Functional analysis of the copper-dependent quercetin 2,3-dioxygenase. 1. Ligand-induced coordination changes probed by X-ray crystallography: inhibition,ordering effect,and mechanistic insights

The crystal structures of the copper-dependent Aspergillus japonicus quercetin 2,3-dioxygenase (2,3QD) complexed with the inhibitors diethyldithiocarbamate (DDC) and kojic acid (KOJ) are reported at 1.70 and 2.15 A resolution, respectively. Both inhibitors asymmetrically chelate the metal center and assume a common orientation in the active site cleft. Their molecular plane blocks access to the inner portion of the cavity which is lined by the side chains of residues Met51, Thr53, Phe75, Phe114, and Met123 and which is believed to bind the flavonol B-ring of the natural substrate. The binding of the inhibitors brings order into the mixed coordination observed in the native enzyme. DDC and KOJ induce a single conformation of the Glu73 side chain, although in different ways. In the presence of DDC, Glu73 is detached from the copper ion with its carboxylate moiety pointing away from the active site cavity. In contrast, when KOJ is bound, Glu73 ligates the Cu ion through its O(epsilon)(1) atom with a monodentate geometry. Compared to the native coordinating conformation, this conformation is approximately 90 degrees rotated about the chi(3) angle. This latter Glu73 conformation is compatible with the presence of a bound substrate.     

Role of a cysteine residue in the active site of ERK and the MAPKK family

Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFbeta-induced AP-1-dependent luciferase expression with respective IC50 values of 0.08 and 0.05 microM. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the alpha,beta-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sgamma of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nzeta of Lys114, backbone C=O of Ser153, Ndelta2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKalpha/beta/gamma/delta which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

Crystal structure of cold-active protein-tyrosine phosphatase from a psychrophile, Shewanella sp

The cold-active protein-tyrosine phosphatase (CAPTPase) of a psychrophile, Shewanella sp., shows high catalytic activity below 20 degrees C. The catalytic residue of CAPTPase is histidine, as opposed to the cysteine of known protein-tyrosine phosphatases (PTPases), and the enzyme protein has three amino acid sequences, Asp-Xaa-His, Gly-Asp-Xaa-Xaa-Asp-Arg and Gly-Asn-His-Glu, that are observed in many protein-serine/threonine phosphatases (PS/TPases). We have determined the crystal structures of CAPTPase at 1.82 angstroms and the enzyme bound with a phosphate ion at 1.90 angstroms resolution using X-ray crystallography and the multiple isomorphous replacement method. The final refined models are comprised of 331 amino acid residues, two metal ions, 447 water molecules, and an acetate or phosphate ion in an asymmetric unit. The enzyme protein consists of three beta-sheets, termed Sheet I, Sheet I', and Sheet II, and 14 alpha-helices. The CAPTPase has a different overall structure from known protein-tyrosine phosphatases. The arrangement of two metal ions, a phosphate ion and the adjacent amino acid residues in the catalytic site of CAPTPase is identical to that of PS/TPases. Thus, it was confirmed that the CAPTPase was a novel PTPase with a conformation similar to the catalytic site of PS/TPase. We speculate that the hydrophobic moiety around the catalytic residue of CAPTPase might play an important role in eliciting high activity at low temperature.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Metal search: A computer program that helps design tetrahedral metal-binding sites

We describe a computer program (Metal Search) that helps design tetrahedrally coordinated metal binding sites in proteins of known structure. The program takes as input the backbone coordinates of a protein and outputs lists of four residues that might form tetrahedral sites if wild-type amino acids were replaced by cysteine or histidine. The program also outputs the side chain dihedral angles of the amino acids and the coordinates of the predicted metal ion. The only function evaluated by Metal Search is the ability of side chains to meet simple geometric criteria for formation of a tetrahedral site, but these criteria are sufficient to produce a manageably small list that can then be evaluated by other means. The program has been used in the introduction of zinc binding sites in the designed four-helix bundle protein α 4 and in the B1 domain of streptococcal protein G, and in both cases the tetrahedral coordination of a bound metal ion has been confirmed¹ (Klemba, M., Gardner, K. H., Marino, S., Clarke, N. D., and Regan, L., Nature: Structural Biology 2:368–373, 1995). © 1995 Wiley-Liss, Inc.     

Specificity of Mg2+ binding at the Group II intron branch site

Metal ions play a crucial role in the conformation and splicing activity of Group II introns. Results from 2-aminopurine fluorescence and solution NMR studies suggest that metal ion binding within the branch site region of native D6 of the Group II intron is specific for alkaline earth metal ions and involves inner sphere coordination. Although Mg(2+) and Ca(2+) still bind to a mutant stem loop sequence from which the internal loop had been deleted, ion binding to the mutant RNA results in decreased, rather than increased, exposure of the branch site residue to solvent. These data further support the role of the internal loop in defining branch site conformation of the Group II intron. The specific bound Mg(2+) may play a bivalent role: facilitates the extrahelical conformation of the branch site and has the potential to act as a Lewis acid during splicing.     

Metal substitution of Neurospora copper metallothionein

The binding of diamagnetic Zn(II), Cd(II), and Hg(II) and paramagnetic Co(II) and Ni(II) ions to the apo form of Neurospora metallothionein (MT) was investigated by various spectroscopic techniques. In contrast to native copper MT, which was shown to bind 6 mol of Cu(I)/mol of protein (Lerch, 1980), all substituted forms reveal an overall metal to protein stoichiometry of 3. The charge-transfer (CT) transitions of the complexes containing diamagnetic metal ions as well as the d-d transitions of those with paramagnetic metal ions are indicative of a distorted Td coordination. Electron paramagnetic resonance and absorption measurements of the Co(II) derivative are in agreement with the presence of a metal-thiolate cluster in this protein. Metal titration studies of the apoprotein reveal characteristic spectral features for the derivatives containing two metal equivalents as compared to those with a full complement of three metal ions. The former features are indicative of an exclusive Td type of metal-sulfur coordination whereas the latter suggest that the third metal ion is coordinated in a different fashion. This finding is in agreement with the presence of only seven cysteine residues in Neurospora MT as opposed to nine cysteine residues in the three-metal cluster of the mammalian MT's [Winge, D.R., & Miklossy, K.-A. (1982) J. Biol. Chem. 257, 3471].     

The role of GyrB in the DNA cleavage-religation reaction of DNA gyrase: a proposed two metal-ion mechanism

We have examined the role of the DNA gyrase B protein in cleavage and religation of DNA using site-directed mutagenesis. Three aspartate residues and a glutamate residue: E424, D498, D500 and D502, thought to co-ordinate a magnesium ion, were mutated to alanine; in addition, the glutamate residue and one aspartate residue were mutated to glutamine and asparagine, respectively. We have shown that these residues are important for the cleavage-religation reaction and are likely to be involved in magnesium ion co-ordination. On separate mutation of two of these aspartate residues to cysteine or histidine, the metal ion preference for the DNA relaxation activity of gyrase changed from magnesium to manganese (II). We present evidence to support the idea that cleavage of each DNA strand involves two or more metal ions, and suggest a scheme for the DNA cleavage chemistry of DNA gyrase involving two metal ions.