Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase.

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies "open" and "closed" CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

Crystal structure of the N-terminal domain of the TyrR transcription factor responsible for gene regulation of aromatic amino acid biosynthesis and transport in Escherichia coli K12.

The X-ray structure of the N-terminal domain of TyrR has been solved to a resolution of 2.3 A. It reveals a modular protein containing an ACT domain, a connecting helix, a PAS domain and a C-terminal helix. Two dimers are present in the asymmetric unit with one monomer of each pair exhibiting a large rigid-body movement that results in a hinging around residue 74 of approximately 50 degrees . The structure of the dimer is discussed with reference to other transcription regulator proteins. Putative binding sites are identified for the aromatic amino acid cofactors.

An improved resolution structure of the human beta common receptor involved in IL-3, IL-5 and GM-CSF signalling which gives better definition of the high-affinity binding epitope.

X-ray diffraction has been used to produce and refine a model of the extracellular domains of the beta common cytokine receptor. A minor improvement in resolution has resulted in improved electron-density maps, which have given a clearer indication of the position and stabilization of the key residues Tyr15, Phe79, Tyr347, His349, Ile350 and Tyr403 in the elbow region between domain 1 and domain 4 of the dimer-related molecule.

Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution.

Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences.

Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.

The major signalling entity of the receptors for the haemopoietic cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is the shared beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the beta(c) subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N-terminal sequence. N-glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X-ray crystallographic studies, following mutagenesis to remove one of the N-glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 A and 103.5 A, diffracted to a resolution of 2.9 A using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.

The structure of the PII-ATP complex.

PII is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of PII was solved using a hexagonal crystal form (form I). The more physiologically relevant form of PII is a complex with small molecule effectors. We describe the structure of PII with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of PII with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the PII/ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that PII and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.

Structure of the complete extracellular domain of the common beta subunit of the human GM-CSF, IL-3, and IL-5 receptors reveals a novel dimer configuration.

The receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consist of ligand-specific alpha receptor subunits that play an essential role in the activation of the shared betac subunit, the major signaling entity. Here, we report the structure of the complete betac extracellular domain. It has a structure unlike any class I cytokine receptor described thus far, forming a stable interlocking dimer in the absence of ligand in which the G strand of domain 1 hydrogen bonds into the corresponding beta sheet of domain 3 of the dimer-related molecule. The G strand of domain 3 similarly partners with the dimer-related domain 1. The structure provides new insights into receptor activation by the respective alpha receptor:ligand complexes.

Preliminary X-ray crystallographic and NMR studies on the exonuclease domain of the epsilon subunit of Escherichia coli DNA polymerase III.

The structured core of the N-terminal 3'-5' exonuclease domain of epsilon, the proofreading subunit of Escherichia coli DNA polymerase III, was defined by multidimensional NMR experiments with uniformly (15)N-labeled protein: it comprises residues between Ile-4 and Gln-181. A 185-residue fragment, termed epsilon(1-185), was crystallized by the hanging drop vapor diffusion method in the presence of thymidine-5'-monophosphate, a product inhibitor, and Mn(2+) at pH 5.8. The crystals are tetragonal, with typical dimensions 0.2 mm x 0.2 mm x 1.0 mm, grow over about 2 weeks at 4 degrees C, and diffract X-rays to 2.0 A. The space group was determined to be P4(n)2(1)2 (n = 0, 1, 2, 3), with unit cell dimensions a = 60.8 A, c = 111.4 A.

Structure of the C123S mutant of dienelactone hydrolase (DLH) bound with the PMS moiety of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF).

The structure of DLH (C123S) with PMS bound was solved to 2.5 A resolution (R factor = 15.1%). PMSF in 2-propanol was delivered directly to crystals in drops and unexpectedly caused the crystals to dissolve. New crystals displaying a different morphology emerged within 2 h in situ, a phenomenon that appears to be described for the first time. The changed crystal form reflected altered crystal-packing arrangements elicited by structural changes to the DLH (C123S) molecule on binding inhibitor. The new unit cell remained in the P2(1)2(1)2(1) space group but possessed different dimensions. The structure showed that PMS binding in DLH (C123S) caused conformational changes in the active site and in four regions of the polypeptide chain that contain reverse turns. In the active site, residues with aromatic side chains were repositioned in an edge-to-face cluster around the PMS phenyl ring. Their redistribution prevented restabilization of the triad His202 side chain, which was disordered in electron-density maps. Movements of other residues in the active site were shown to be related to the four displaced regions of the polypeptide chain. Their implied synergy suggests that DLH may be able to accommodate and catalyse a range of compounds unrelated to the natural substrate owing to an inherent coordinated flexibility in its overall structure. Implications for mechanism and further engineering studies are discussed.

Expression, purification and preliminary crystallographic studies of a hyperthermophilic esterase from Archaeoglobus fulgidus.

An esterase from the hyperthermophilic archeon Archaeoglobus fulgidus has been expressed, purified and crystallized in a form suitable for structure analysis. The enzyme has a molecular mass of 35 467 Da and shows sequence similarity to other esterases known to possess the alpha/beta hydrolase fold. The crystals diffract to 2.8 A and belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 155.6, b = 155.0, c = 162.4 A.

The Escherichia coli signal transducers PII (GlnB) and GlnK form heterotrimers in vivo: fine tuning the nitrogen signal cascade.

The PII protein is Escherichia coli's cognate transducer of the nitrogen signal to the NRII (NtrB)/NRI (NtrC) two-component system and to adenylyltransferase. Through these two routes, PII regulates both amount and activity of glutamine synthetase. GlnK is the recently discovered paralogue of PII, with a similar trimeric x-ray structure. Here we show that PII and GlnK form heterotrimers, in E. coli grown in nitrogen-poor medium. In vitro, fully uridylylated heterotrimers of the two proteins stimulated the deadenylylation activity of adenylyltransferase, albeit to a lower extent than homotrimeric PII-UMP. Fully uridylylated GlnK did not stimulate, or hardly stimulated, the deadenylylation activity. We propose that uridylylated PII/GlnK heterotrimers fine-regulate the activation of glutamine synthetase. The PII/GlnK couple is a first example of prokaryotic signal transducer that can form heterotrimers. Advantages of hetero-oligomer formation as molecular mechanism for fine-regulation of signal transduction are discussed.

Crystallization of the N-terminal domain of the Escherichia coli regulatory protein TyrR.

The N-terminal domain of the regulatory protein TyrR from Escherichia coli forms a dimer in solution and has been purified and crystallized. The crystals belong to space group C2 with unit-cell parameters a = 134.5, b = 72.1, c = 96.7 A, beta = 98.5 degrees. The crystals diffract to 2.8 A. Assuming a molecular weight of 23219 Da, a V(m) of 2.5 A(3) Da(-1) is obtained for two dimers in the asymmetric unit.

Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase.

BACKGROUND: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. RESULTS: The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. CONCLUSIONS: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.

Of barn owls and bankers: a lush variety of alpha/beta hydrolases.

alpha/beta Hydrolase fold proteins are an important, diverse, widespread group of enzymes not yet fully exploited by structural biologists. We describe the current state of knowledge of this family, and suggest a smaller definition of the required core and some possible future avenues of exploration.

Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction.

BACKGROUND: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. RESULTS: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 A resolution. The core structure is homologous to AND-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP+, hydrogen bonding to the catalytic His225 as well as obstructing access of the C4 acid substrate. Two loops flanking the active site, termed the Arg2 and Trp loops, that contain the C4 acid substrate binding residues are prevented from closing by the C-terminal extension. CONCLUSIONS: The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP+ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP+ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP+ but not NADPH.

Crystallization and preliminary crystallographic studies of chloroplast NADP-dependent malate dehydrogenase from Flaveria bidentis.

Crystals of chloroplast NADP-dependent malate dehydrogenase have been grown both with and without the cofactor NADP present. The enzyme has a molecular weight of 43 kDa per subunit and exists as a dimer in solution. The crystals diffract to 2.8 A and belong to the space group P3221 with cell dimensions a = 148.1, c = 65.5 A.

Crystallization and preliminary X-ray analysis of IND, an enzyme with indole oxygenase activity from Chromobacterium violaceum.

IND, a redox flavoprotein from Chromobacterium violaceum has been crystallized in the presence and absence of NADH. The crystals belong to the space group P41212 or its enantiomorph P43212 with a = 73.9 and c = 153.6 A. There is one molecule per asymmetric unit and the crystals diffract beyond 2.1 A resolution.

Crystallization and preliminary X-ray analysis of Escherichia coli GlnK.

The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.

GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition.

GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria. PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins.

The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains.

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.