Matthew M. Benning

Crystallization and structure determination to 2.5-A resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120.

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.

The Binding of Substrate Analogs to Phosphotriesterase

Phosphotriesterase (PTE) fromPseudomonas diminuta catalyzes the detoxification of organophosphates such as the widely utilized insecticide paraoxon and the chemical warfare agent sarin. The three-dimensional structure of the enzyme is known from high resolution x-ray crystallographic analyses. Each subunit of the homodimer folds into a so-called TIM barrel, with eight strands of parallel β-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the β-barrel. Here, we describe the three-dimensional structure of PTE complexed with the inhibitor diisopropyl methyl phosphonate, which serves as a mimic for sarin. Additionally, the structure of the enzyme complexed with triethyl phosphate is also presented. In the case of the PTE-diisopropyl methyl phosphonate complex, the phosphoryl oxygen of the inhibitor coordinates to the more solvent-exposed zinc ion (2.5 Å), thereby lending support to the presumed catalytic mechanism involving metal coordination of the substrate. In the PTE-triethyl phosphate complex, the phosphoryl oxygen of the inhibitor is positioned at 3.4 Å from the more solvent-exposed zinc ion. The two structures described in this report provide additional molecular understanding for the ability of this remarkable enzyme to hydrolyze such a wide range of organophosphorus substrates.

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2·5 Å resolution

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.

The structure of carbamoyl phosphate synthetase determined to 2.1 Å resolution

Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxyphosphate and carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

Divergence of Chemical Function in the Alkaline Phosphatase Superfamily: Structure and Mechanism of the P-C Bond Cleaving Enzyme Phosphonoacetate Hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution.

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95A resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.

The Structure of Classical Swine Fever Virus Npro: A Novel Cysteine Autoprotease and Zinc-Binding Protein Involved in Subversion of Type I Interferon Induction

Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (Npro) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, Npro is no longer active as a protease. The released Npro suppresses the induction of the hosts type-I interferon-α/β (IFN-α/β) response. Npro binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus Npro. Npro is structurally distinct from other known cysteine proteases and has a novel “clam shell” fold consisting of a protease domain and a zinc-binding domain. The unique fold of Npro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of Npro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of Npros interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of Npro, and its role in subversion of host immune response.

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30

The existence of N‐formylated sugars in the O‐antigens of Gram‐negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose‐1‐phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N‐formyltransferases that utilize N10‐formyltetrahydrofolate as the carbon source. Here we describe the three‐dimensional structure of one of these N‐formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP‐4‐amino‐4,6‐dideoxyglucose (dTDP‐Qui4N) to dTDP‐4,6‐dideoxy‐4‐formamido‐d‐glucose (dTDP‐Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP‐Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β‐sheet flanked on one side by three α‐helices and on the other side by mostly random coil. This N‐terminal domain is followed by an α‐helix and a β‐hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent‐exposed. Notably, the pyranosyl moiety of dTDP‐Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N‐formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP‐sugar substrate.

Crystal structure of Npun_R1517, a putative negative regulator of heterocyst differentiation from Nostoc punctiforme PCC 73102

Cyanobacteria are ancient photosynthetic bacteria ( 2.85 billion years old1) that are capable of fixing atmospheric N2 during periods of nitrogen starvation in their growing environment.2 Among the five distinct classes of cyanobacteria, filamentous cyanobacteria such as Nostoc punctiforme strain PCC 73102 form chains of actively dividing vegetative cells capable of carrying out photosynthesis.2 Within 18–24 h after the onset of nitrogen starvation, 10% of vegetative cells, evenly spaced along the filament, differentiate into ‘‘heterocysts,’’2,3 specialized cells which have a thick glycolipid-rich cellular wall that helps maintain a microaerobic environment required for function of nitrogenases involved in nitrogen fixation.4 Heterocyst differentiation appears to be regulated by a primitive, yet complex, intercellular regulatory network involving hundreds of genes.5 Several genes have been determined to play a specific role in heterocyst differentiation, including two key transcriptional regulators: nitrogen control protein A (NtcA) that manages global nitrogen uptake,6 and HetR, an autoregulatory DNAbinding protein considered to be a master regulator of heterocyst differentiation.7,8 Heterocyst differentiation is also regulated by PatS, which appears to be an intercellular signaling peptide produced by developing heterocysts to inhibit differentiation of neighboring vegetative cells.9–11 Certain genes of yet unknown biochemical function have been shown to suppress heterocyst differentiation when overexpressed in wild-type cells, including HetY12 and HetN,13 while overexpression of HetL, a pentapeptide repeat protein whose structure has recently been solved,14 has been shown to stimulate heterocyst differentiation.15 Asr1734 from Nostoc sp. strain PCC 7120 (also referred to as Anabaena sp. strain PCC 7120) has also recently been reported to be a negative regulator of heterocyst differentiation.16 Asr1734 is a small protein of 93 residues (MW 10.75 kDa) that appears to be an orphan with homologs only in other filamentous cyanobacteria: Nostoc punctiforme PCC 73102 (Npun_R1517, 91% seq. id.), Nodularia spumigena CCY9414 (N9414_00595, 93% seq. id.), and Anabaena variabilis (AvA0302, 100% seq. id.). As a first step toward investigating its biochemical function, we have determined the crystal structure of Npun_R1517 from Nostoc punctiforme PCC 73102, at 2.0 Å resolution.