Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2

Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.     

Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.     

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.     

Archaeoglobus fulgidus CopB is a thermophilic Cu2+-ATPase: functional role of its histidine-rich-N-terminal metal binding domain

P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 degrees C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 microm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates.     

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.     

X-Ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes

Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge x-ray absorption fine-structure spectroscopy, we report here on the local structure of Zn2+ bound stoichiometrically to noncrystallized cyt bc1 complexes. We performed a comparative x-ray absorption fine-structure spectroscopy study by examining avian, bovine, and bacterial enzymes. A large number of putative clusters, built by combining information from first-shell analysis and metalloprotein databases, were fitted to the experimental spectra by using ab initio simulations. This procedure led us to identify the binding clusters with high levels of confidence. In both the avian and bovine enzyme, a tetrahedral ligand cluster formed by two His, one Lys, and one carboxylic residue was found, and this ligand attribution fit the crystallographic Zn01 location of the avian enzyme. In the chicken enzyme, the ligands were the His121, His268, Lys270, and Asp253 residues, and in the homologous bovine enzyme they were the His121, His267, Lys269, and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of 6. The best-fit octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue, and two water molecules. It was interesting that by aligning the crystallographic structures of the bacterial and avian enzymes, this group of residues was found located in the region homologous to that of the Zn01 site. This cluster included the His276, Asp278, Glu295, and Asn279 residues of the cyt b subunit. The conserved location of the Zn2+ binding sites at the entrance of the putative proton release pathways, and the presence of His residues point to a common mechanism of inhibition. As previously shown for the photosynthetic bacterial reaction center, zinc would compete with protons for binding to the His residues, thus impairing their function as proton donors/acceptors.     

Bacterial transition metal P(1B)-ATPases: transport mechanism and roles in virulence

P(1B)-type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This paper reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu(+)-ATPases are the most frequently observed and best-characterized members of this group of transporters. However, bacterial genomes show diverse arrays of P(1B)-type ATPases with a range of substrates (Cu(+), Zn(2+), Co(2+)). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport nonphysiological substrates (Cd(2+), Pb(2+), Au(+), Ag(+)). P(1B)-type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P(1B)-type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P(1B)-ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P(1B)-ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.     

X-ray absorption studies of Zn-binding sites in Escherichia coli transhydrogenase and its βH91K mutant

Transhydrogenase couples hydride transfer between NADH and NADP⁺ to proton translocation across a membrane. The binding of Zn²⁺ to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn²⁺ bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which βHis91 was replaced by Lys (βH91K). This well-conserved His residue, located in the membrane-spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn²⁺. The XAFS analysis has identified a Zn²⁺-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn²⁺ inhibits proton translocation by competing with H⁺ binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and βH91K. Evidently, βHis91 is not directly involved in Zn²⁺ binding. The locus of βHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.     

Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-A resolution: identification of the active site and possible catalytic residues

The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS)     

The MntC crystal structure suggests that import of Mn2+ in cyanobacteria is redox controlled

The MntC protein is the periplasmic solute-binding protein component of the high-affinity manganese ATP-binding cassette-type transport system in the cyanobacterium Synechocytis PCC sp. 6803. We have determined the structure of recombinant MntC at 2.9 A resolution by X-ray crystallography using a combination of multi-wavelength anomalous diffraction and molecular replacement. The presence of Mn2+ in the metal ion-binding site was ascertained by use of anomalous difference electron density maps using diffraction data collected at the Mn absorption edge. The MntC protein is similar to previously determined metal ion-binding, solute-binding proteins with two globular domains connected by an extended alpha-helix. However, the metal ion-binding site is asymmetric, with two of the four ligating residues (Glu220 and Asp295) situated closer to the ion than the two histidine residues (His89 and His154). A unique characteristic of the MntC is the existence of a disulfide bond between Cys219 and Cys268. Analysis of amino acid sequences of homologous proteins shows that conservation of the cysteine residues forming the disulfide bond occurs only in cyanobacterial manganese solute-binding proteins. One of the monomers in the MntC asymmetric unit trimer is disordered significantly in the globular domain containing the disulfide bond. The electron density on the manganese ion and on the disulfide bond in this monomer indicates that reduction of this bond changes the relative position of the lower domain and of the Glu220 ligand, potentially lowering the affinity towards Mn2+. This is confirmed by reduction of the disulfide bond in vitro, showing the release of bound Mn2+. We propose that the reduction or oxidation state of the disulfide bond can alter the binding affinity of the protein towards Mn2+ and thus determine whether these ions will be transported into the cytoplasm, or be available for photosystem II biogenesis in the periplasm.     

Characterization of the zinc binding activity of the rubella virus nonstructural protease

The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463-4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn(65) binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV-CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.     

Activation of Archaeoglobus fulgidus Cu(+)-ATPase CopA by cysteine

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu(+) across the cell membrane. Millimolar concentration of Cys dramatically increases ( congruent with 800%) the activity of CopA and other P(IB)-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal ( congruent with 1 microM) together with the low Cu(+)-Cys K(D) (<10(-10)M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu(+) chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu(+), suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K(m) suggesting no changes in the E(1)<-->E(2) equilibrium. Characterization of Cu(+) transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu(+) can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu(+)-Cys complex.     

Zn(II) coordination domain mutants of T4 gene 32 protein.

Gene 32 protein (g32P), the replication accessory single-stranded nucleic acid binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. Zinc coordination provides structural stability to the DNA-binding core domain of the molecule, termed g32P-(A+B) (residues 22-253). Optical absorption studies with the Co(II)-substituted protein and 113Cd NMR spectroscopy of 113Cd(II)-substituted g32P-(A+B) show that the metal coordination sphere in g32P is characterized by approximately tetrahedral ligand symmetry and ligation by the Cys-S- atoms of Cys77, Cys87, and Cys90. These studies predicted the involvement of a fourth protein-derived non-thiol ligand to complete the tetrahedral complex, postulated to be His81 on the basis of primary structure prediction and modeling [Giedroc, D.P., Johnson, B.A., Armitage, I.M., & Coleman, J.E. (1989) Biochemistry 28, 2410-2418]. To test this model, we have employed site-directed mutagenesis to substitute each of the two histidine residues in g32P (His64 and His81), accompanied by purification and structural characterization of these single-site mutant proteins. We show that g32P's containing any of three substitutions at residue 64 (H64Q, H64N, and H64L) are isolated from Escherichia coli in a Zn(II)-free form [less than or equal to 0.03 g.atom Zn(II)]. All derivatives show extremely weak affinity for the ssDNA homopolymer poly(dT). All are characterized by a far-UV-CD spectrum reduced in negative intensity relative to the wild-type protein. These structural features parallel those found for the known metal ligand mutant Cys87----Ser87 (C87S) g32P. In contrast, g32P-(A+B) containing a substitution of His81 with glutamine (H81Q), alanine (H81A) or cysteine (H81C), contains stoichiometric Zn(II) as isolated and binds to polynucleotides with an affinity comparable to the wild-type g32P-(A+B). Spin-echo 1H NMR spectra recorded for wild-type and H81Q g32P-(A+B) as a function of pH allow the assignment of His81 ring proteins to delta = 6.81 and 6.57 ppm, respectively, at pH 7.8, corresponding to the C and D histidyl protons of 1H-His-g32P-(A+B) [Pan, T., Giedroc, D.P., & Coleman, J.E. (1989) Biochemistry 28, 8828-8832]. These resonances shift downfield as the pH is reduced from 7.8 to 6.6 without metal dissociation, a result incompatible with His81 donating a ligand to the Zn(II) in wild-type g32P. Likewise, Cys81 in Zn(II) H81C g32P is readily reactive with 5,5'-dithiobis(2-nitrobenzoic acid), unlike metal ligands Cys77, Cys87, and Cys90.(ABSTRACT TRUNCATED AT 400 WORDS)     

The crystal structure of metal-free human EF-hand protein S100A3 at 1.7-A resolution

S100A3 is a unique member of the EF-hand superfamily of Ca(2+)-binding proteins. It binds Ca(2+) with poor affinity (K(d) = 4-35 mm) but Zn(2+) with exceptionally high affinity (K(d) = 4 nm). This high affinity for Zn(2+) is attributed to the unusual high Cys content of S100A3. The protein is highly expressed in fast proliferating hair root cells and astrocytoma pointing toward a function in cell cycle control. We determined the crystal structure of the protein at 1.7 A. The high resolution structure revealed a large distortion of the C-terminal canonical EF-hand, which most likely abolishes Ca(2+) binding. The crystal structure of S100A3 allows the prediction of one putative Zn(2+) binding site in the C terminus of each subunit of S100A3 involving Cys and His residues in the coordination of the metal ion. Zn(2+) binding induces a large conformational change in S100A3 perturbing the hydrophobic interface between two S100A3 subunits, as shown by size exclusion chromatography and CD spectroscopy.