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A copper-containing amine oxidase (PPLO) from the yeast Pichia pastoris has been purified and crystallized in two forms. PPLO is a glycoprotein. The molecular mass from SDS-polyacrylamide gels is 112 kDa, consistent with 20% glycosylation by weight (the calculated molecular weight of the polypeptide is 89.7 kDa). Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 163.7, b = 316.1, c = 84.0 A, diffract to 2.65 A resolution. Monoclinic crystals belonging to space group C2, with unit-cell parameters a = 248.4, b = 121.1, c = 151.8 A, beta = 124.6 degrees, diffract to 1.65 A resolution. Native data have been recorded from each crystal form at 100 K using synchrotron radiation. A self-rotation function for the monoclinic crystal form reveals the presence of a non-crystallographic twofold axis perpendicular to the crystallographic twofold axis, consistent with the presence of two dimers in the asymmetric unit.

SoxAX cytochromes catalyze the formation of heterodisulfide bonds between inorganic sulfur compounds and a carrier protein, SoxYZ. They contain unusual His/Cys-ligated heme groups with complex spectroscopic signatures. The heme-ligating cysteine has been implicated in SoxAX catalysis, but neither the SoxAX spectroscopic properties nor its catalysis are fully understood at present. We have solved the first crystal structure for a group 2 SoxAX protein (SnSoxAX), where an N-terminal extension of SoxX forms a novel structure that supports dimer formation. Crystal structures of SoxAX with a heme ligand substitution (C236M) uncovered an inherent flexibility of this SoxA heme site, with both bonding distances and relative ligand orientation differing between asymmetric units and the new residue, Met(236), representing an unusual rotamer of methionine. The flexibility of the SnSoxAX(C236M) SoxA heme environment is probably the cause of the four distinct, new EPR signals, including a high spin ferric heme form, that were observed for the enzyme. Despite the removal of the catalytically active cysteine heme ligand and drastic changes in the redox potential of the SoxA heme (WT, -479 mV; C236M, +85 mV), the substituted enzyme was catalytically active in glutathione-based assays although with reduced turnover numbers (WT, 3.7 s(-1); C236M, 2.0 s(-1)). SnSoxAX(C236M) was also active in assays using SoxYZ and thiosulfate as the sulfur substrate, suggesting that Cys(236) aids catalysis but is not crucial for it. The SoxYZ-based SoxAX assay is the first assay for an isolated component of the Sox multienzyme system.

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2'-(or -3')-O-(N-methylanthraniloyl)-beta,gamma-imidoguanosine 5'-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K(+), which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K(+) coordination, and mutation of this residue abolished K(+)-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K(+)-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

Vacuolar-type ATPases (V-ATPases) exist in various cellular membranes of many organisms to regulate physiological processes by controlling the acidic environment. Here, we have determined the crystal structure of the A(3)B(3) subcomplex of V-ATPase at 2.8 A resolution. The overall construction of the A(3)B(3) subcomplex is significantly different from that of the alpha(3)beta(3) sub-domain in F(o)F(1)-ATP synthase, because of the presence of a protruding 'bulge' domain feature in the catalytic A subunits. The A(3)B(3) subcomplex structure provides the first molecular insight at the catalytic and non-catalytic interfaces, which was not possible in the structures of the separate subunits alone. Specifically, in the non-catalytic interface, the B subunit seems to be incapable of binding ATP, which is a marked difference from the situation indicated by the structure of the F(o)F(1)-ATP synthase. In the catalytic interface, our mutational analysis, on the basis of the A(3)B(3) structure, has highlighted the presence of a cluster composed of key hydrophobic residues, which are essential for ATP hydrolysis by V-ATPases.

G proteins are key molecular switches in the regulation of membrane protein function and signal transduction. The prokaryotic membrane protein FeoB is involved in G protein coupled Fe(2+) transport, and is unique in that the G protein is directly tethered to the membrane domain. Here, we report the structure of the soluble domain of FeoB, including the G protein domain, and its assembly into an unexpected trimer. Comparisons between nucleotide free and liganded structures reveal the closed and open state of a central cytoplasmic pore, respectively. In addition, these data provide the first observation of a conformational switch in the nucleotide-binding G5 motif, defining the structural basis for GDP release. From these results, structural parallels are drawn to eukaryotic G protein coupled membrane processes.

The acid-induced arginine decarboxylase is part of an enzymatic system in Escherichia coli that contributes to making this organism acid resistant. The arginine decarboxylase is a vitamin B(6)-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced arginine decarboxylase by X-ray crystallography to 2.4 A resolution. The arginine decarboxylase structure revealed a ca. 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevents inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.3 kDa). Apo-CopK associates in solution to form a dimer (K(D) approximately 10(-5) M) whose structure was defined by NMR and X-ray crystallography. The individual molecules feature two antiparallel beta-sheets arranged in a sandwich-like structure and interact through C-terminal beta-strands. It binds Cu(II) with low affinity (K(D)(Cu(II)) > 10(-6) M) but Cu(I) with high affinity (K(D)(Cu(I)) = 2 x 10(-11) M). Cu(I)-CopK was also a dimer in the solid state and featured a distorted tetrahedral site Cu(I)(S-Met)(3)(NCS). The isothiocyanato ligand originated from the crystallization solution. Binding of Cu(I) or Ag(I), but not of Cu(II), favored the monomeric form in solution. While Ag(I)-CopK was stable as isolated, Cu(I)-CopK was moderately air-sensitive due to a strong binding cooperativity between Cu(I) and Cu(II). This was documented by determination of the Cu(I) and Cu(II) binding affinities in the presence of the other ion: K(D)(Cu(I)) = 2 x 10(-13) M and K(D)(Cu(II)) = 3 x 10(-12) M, that is, binding of Cu(II) increased the affinity for Cu(I) by a factor of approximately 10(2) and binding of Cu(I) increased the affinity for Cu(II) by a factor of at least 10(6). Stable forms of both Cu(I)Cu(II)-CopK and Ag(I)Cu(II)-CopK were isolated readily. Consistent with this unprecedented copper binding chemistry, NMR spectroscopy detected three distinct forms: apo-CopK, Cu(I)-CopK and Cu(I)Cu(II)-CopK that do not exchange on the NMR time scale. This information provides a valuable guide to the role of CopK in copper resistance.

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as "mixed disulfide fishing" in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5 angstrom) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes. (c) 2008 Elsevier Ltd. All rights reserved.

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.

Escherichia coli dihydroorotase has been crystallized in the presence of the product, L-dihydroorotate (L-DHO), and the structure refined at 1.9A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-D,L-aspartate (D, L-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, L-CA-asp bound at the active site and the other having L-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105-115 has different conformations in the two subunits. In the case of the L-CA-asp-bound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the L-DHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of L-DHO to L-CA-asp at low concentrations of L-DHO shows positive cooperativity with a Hill coefficient n=1.57(+/-0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256-His257-Arg258, near the subunit interface.

Aminopeptidase P (APPro) is a metalloprotease whose active site includes a dinuclear manganese(II) cluster. The enzyme cleaves the N-terminal residue from a polypeptide when the second residue is proline. A complex of Escherichia coli APPro (EcAPPro) with an inhibitor, apstatin [N-(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl-L-prolyl-L-prolyl-L-alaninamide], has been crystallized. Apstatin binds to the active site of EcAPPro with its N-terminal amino group coordinated to one of the two Mn(II) atoms at the metal centre. The apstatin hydroxyl group replaces a hydroxide ion which bridges the two metal atoms in the native enzyme. The first proline residue of apstatin lies in a small hydrophobic cleft. The structure of the apstatin-EcAPPro complex has been refined at 2.3 A resolution with residuals R = 0.179 and R(free) = 0.204. The structure of the complex illustrates how apstatin inhibits APPro and suggests how substrates may bind to the enzyme, but the basis of the proline-specificity remains elusive.

The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781-9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of two functionally related enzymes, aminopeptidase P (APPro) and creatinase. In addition, the catalytic C-terminal domain of Pfprol has a polypeptide fold resembling that of the sole domain of a fourth enzyme, methionine aminopeptidase (MetAP). The active sites of APPro and MetAP, like that of Pfprol, include a dinuclear metal center. The metal ligands in the three enzymes are homologous. Comparisons with the molecular structures of APPro and MetAP suggest how Pfprol discriminates against oligopeptides and in favor of Xaa-Pro substrates. The crystal structure of Pfprol was solved by multiple-wavelength anomalous dispersion. The crystals yielded diffraction data of relatively high quality and resolution, despite the fact that one of the two protein subunits in the asymmetric unit was found to be significantly disordered. The final R and R(free) values are 0.24 and 0.28, respectively.

LMO4 is the most recently discovered member of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. LMO4 is comprised primarily of two tandemly repeated LIM domains and interacts with the ubiquitous nuclear adaptor protein ldb1. This interaction is mediated via the LIM domains of LMO4 and the LIM-interaction domain (LID) of ldb1. An intramolecular complex, termed FLINC4, consisting of the two LIM domains from LMO4 linked to the LID domain of ldb1 via a flexible linker has been engineered, purified and crystallized. The trigonal crystals, which belong to space group P312 with unit-cell parameters a = 61.3, c = 93.2 A, diffract to 1.3 A resolution and contain one molecule of FLINC4 per asymmetric unit. Native and multiple-wavelength anomalous dispersion (MAD) data collected at the Zn X-ray absorption edge have been recorded to 1.3 and 1.7 A resolution, respectively. Anomalous Patterson maps calculated with data collected at the peak wavelength show strong peaks sufficient to determine the positions of four Zn atoms per asymmetric unit.

Dihydroorotase (DHOase) catalyses the formation of L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway. The type I DHOase domain from hamster forms part of the trifunctional enzyme CAD. The hamster DHOase domain has been cloned and expressed in Escherichia coli. Solutions of the homodimeric protein convert to a homotetrameric species when incubated at ambient temperature. Formation of the tetrameric species is mediated via disulfide linkages between single free cysteine residues on the surface of each monomer. This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer. The crystallization of pure tetrameric DHOase results in two crystal forms: form I, with space group C222(1) and unit-cell parameters a = 127.1, b = 603.5, c = 144.7 A, and form II, with space group P2(1) and unit-cell parameters a = 260.5, b = 148.2, c = 308.0 A, beta = 102.2 degrees. Data have been recorded to 4.3 and 4.0 A resolution, respectively.

Selenate reductase from Thauera selenatis was crystallized using ammonium sulfate as a precipitant. Crystals of selenate reductase belong to the space group C2, with unit-cell parameters a = 116.9, b = 67.5, c = 186.7 A, beta = 90 degrees. Native data to 2.1 A resolution have been collected and a heavy-atom derivative has been identified following soaking of the crystals in a solution of trimethyl lead acetate.

The prolidase (proline-specific amino dipeptidase) from the hyperthermophilic archaeon Pyrococcus furiosus has been crystallized. The enzyme has been shown to be a homodimer and to require two Co atoms per subunit for optimum activity. Two crystal forms have been obtained under similar growth conditions. Both are monoclinic, space group P2(1). Form I has unit-cell parameters a = 130.4, b = 97.4, c = 129.9 A, beta = 118.3 degrees. Form II has a smaller unit cell, with a = 56.5, b = 97.3, c = 70.0 A, beta = 97.1 degrees. If the crystal density is assumed to lie near the center of the normal range then the form I crystals will have four dimers per asymmetric unit, whereas the form II crystals will have only one dimer in each asymmetric unit. Diffraction data have been recorded from native form I and form II crystals to resolutions of 3.2 and 1.95 A, respectively.

Eleven mutant forms of the ferredoxin from Clostridium pasteurianum (CpFd; 2 Fe4S4; 6200 Da) have been isolated in which six surface carboxylates are changed systematically to their uncharged but stereochemically equivalent carboxamide analogues. Such changes provide molecules which vary in overall charge and its surface distribution but vary minimally in structure and reduction potential. Glu-17 and Asp-6, -27, -33, -35, and -39 were converted providing six single mutants, four double mutants and one triple mutant. The proteins were characterised by UV-visible spectroscopy, square-wave voltammetry and 1H NMR. Their ability to mediate electron transfer between spinach NADH:ferredoxin oxidoreductase and horse heart cytochrome c was assessed. Each mutant is 30-100% as active as the recombinant protein with the triple mutant D33,35,39N being least active. Second-order rate constants k2 for the oxidation of reduced mutant ferredoxins by [Co(NH3)6]3+ were measured at 25 degrees C and I = 0.1 M by stopped-flow techniques. Each mutant displayed saturation kinetics with k2 being 30-100% of that for the recombinant protein. The rates were moderately sensitive to ionic strength. Variation in association constant K could not be detected within the confidence limits of the data. Overall the effects of the mutations were minor. In contrast to human and Anabaena 7120 [Fe2S2]-ferredoxins, electron transfer does not appear to rely on the presence of one or two specific surface carboxylate residues. It may occur from multiple sites on the surface of CpFd with recognition processes for its many physiological redox partners being controlled by relative reduction potentials, in addition to unidentified criteria. The conclusions are consistent with previous results for another series of mutant CpFd proteins interacting with physiological redox partners pyruvate: Fd oxidoreductase and hydrogenase (J.M. Moulis, V. Davasse (1995) Biochemistry 34, 16781-16788).

The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood. In particular, it is not yet known how the pivotal step in GTP hydrolysis is achieved: alignment of a catalytic water molecule. In the current study, the crystal structure of the soluble domains from Streptococcus thermophilus FeoB (NFeoB(St)) in complex with the activating potassium ion and a transition-state analogue, GDP⋅AlF(4) (-), reveals a novel mode of water alignment involving contacts with the protein backbone only. In parallel to the structural studies, a series of seven mutant proteins were constructed that targeted conserved residues at the active site of NFeoB(St), and the nucleotide binding and hydrolysis properties of these were measured and compared to the wild-type protein. The results show that mutations in Thr35 abolish GTPase activity of the protein, while other conserved residues (Tyr58, Ser64, Glu66 and Glu67) are not required for water alignment by NFeoB(St). Together with the crystal structure, the findings suggest a new mechanism for hydrolysis initiation in small G-proteins, in which the attacking water molecule is aligned by contacts with the protein backbone only.

Dihydroorotase (DHOase) is a zinc metalloenzyme that catalyses the reversible cyclization of N-carbamyl-L-aspartate (CA-asp) to dihydroorotate (DHO) in the de novo pyrimidine biosynthesis. The first structure of a DHOase (from E. coli) has been reported to a resolution of 1.7 A with one homodimer per asymmetric unit [1]. We have collected data from crystals of E. coli DHOase grown in the presence of product, DHO and refined the structure to 1.9 A resolution [2]. As in the original report [1], we find the product DHO bound in the active site of one subunit and CA-asp in the active site of the other. Importantly, we have resolved the conformations of residues B109-112, which were disordered in the reported structure. These residues comprise a loop which takes on different orientations in the two subunits, depending on whether DHO or CA-asp is present in the active site. This is accompanied by movements of residues A/B256258 which seem to 'communicate' between subunits the respective contents of the active sites. Subsequent kinetic analysis at low DHO concentrations shows positive cooperativity [2]. Aspects of this structure of DHOase will be discussed. In addition, the structures of inhibitor complexes and site-directed mutagenesis that allow us to understand more about this loop movement will be described in relation to the enzyme mechanism.

In our recent article Maher et al. (2004) we omitted to cite the work of Meyer et al. (1997) who were the first to construct an Fe2S2 centre in rubredoxin. This work was later extended in Cross et al. (2002), with the construction of other altered metal sites.

The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped-flow kinetic analyses reveal uncoupled nucleotide affinity and release rates, supporting a model where G5 loop movement promotes nucleotide release.