Structural and functional analysis of AsbF: origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis.

Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell.

Protein functional surfaces: global shape matching and local spatial alignments of ligand binding sites.

Background: Protein surfaces comprise only a fraction of the total residues but are the most conserved functional features of proteins. Surfaces performing identical functions are found in proteins absent of any sequence or fold similarity. While biochemical activity can be attributed to a few key residues, the broader surrounding environment plays an equally important role.

Structural insight into acute intermittent porphyria.

Acute intermittent porphyria (AIP), an inherited disease of heme biosynthesis, is one of the most common types of porphyria. Reduced activity of the enzyme porphobilinogen deaminase (PBGD), which catalyzes the sequential condensation of 4 molecules of porphobilinogen to yield preuroporphyrinogen, has been linked to the symptoms of AIP. We have determined the 3-dimensional structure of human PBGD at 2.

Structure of RapA, a Swi2/Snf2 protein that recycles RNA polymerase during transcription.

RapA, as abundant as sigma70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. We report a structure of RapA that is also a full-length structure for the entire Swi2/Snf2 family.

The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues.

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands.

Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza H5N1 virus.

The recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health. Elucidation of the underlying mechanisms of viral replication is critical for development of anti-influenza virus drugs. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer has crucial roles in viral RNA replication and transcription.

Structure of the influenza virus A H5N1 nucleoprotein: implications for RNA binding, oligomerization, and vaccine design.

The threat of a pandemic outbreak of influenza virus A H5N1 has become a major concern worldwide. The nucleoprotein (NP) of the virus binds the RNA genome and acts as a key adaptor between the virus and the host cell. It, therefore, plays an important structural and functional role and represents an attractive drug target.

Crystal structures of the effector-binding domain of repressor Central glycolytic gene Regulator from Bacillus subtilis reveal ligand-induced structural changes upon binding of several glycolytic intermediates.

Expression of genes in the gapA operon encoding five enzymes for triose phosphate interconversion in Bacillus subtilis is negatively regulated by the Central glycolytic genes Regulator (CggR). CggR belongs to the large SorC/DeoR family of prokaryotic transcriptional regulators, characterized by an N-terminal DNA-binding domain and a large C-terminal effector-binding domain. When no glucose is present in growth media, CggR binds to its target DNA sequence and blocks the transcription of genes in the gapA operon.

Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis.

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine.

Unusual binding mode of the 2S4R stereoisomer of the potent aldose reductase cyclic imide inhibitor fidarestat (2S4S) in the 15 K crystal structure of the ternary complex refined at 0.78 A resolution: implications for the inhibition mechanism.

The structure of human aldose reductase in complex with the 2 S4 R stereoisomer of the potent inhibitor Fidarestat ((2 S,4 S)-6-fluoro-2',5'-dioxospiro-[chroman-4,4'-imidazoline]-2-carboxamide) was determined at 15 K and a resolution of 0.78 A. The structure of the complex provides experimental evidence for the inhibition mechanism in which Fidarestat is initially bound neutral and then becomes negatively charged by donating the proton at the 1'-position nitrogen of the cyclic imide ring to the N2 atom of the catalytic His110.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase.

We present results of combined studies of the enzyme human aldose reductase (h-AR, 36 kDa) using single-crystal x-ray data (0.66 A, 100K; 0.80 A, 15K; 1.

The 1.38 A crystal structure of DmsD protein from Salmonella typhimurium, a proofreading chaperone on the Tat pathway.

The DmsD protein is necessary for the biogenesis of dimethyl sulphoxide (DMSO) reductase in many prokaryotes. It performs a critical chaperone function initiated through its binding to the twin-arginine signal peptide of DmsA, the catalytic subunit of DMSO reductase. Upon binding to DmsD, DmsA is translocated to the periplasm via the so-called twin-arginine translocation (Tat) pathway.

Structures of open (R) and close (T) states of prephenate dehydratase (PDT)--implication of allosteric regulation by L-phenylalanine.

The enzyme prephenate dehydratase (PDT) converts prephenate to phenylpyruvate in L-phenylalanine biosynthesis. PDT is allosterically regulated by L-Phe and other amino acids. We report the first crystal structures of PDT from Staphylococcus aureus in a relaxed (R) state and PDT from Chlorobium tepidum in a tense (T) state.

Chapter 4. Predicting and characterizing protein functions through matching geometric and evolutionary patterns of binding surfaces.

Predicting protein functions from structures is an important and challenging task. Although proteins are often thought to be packed as tightly as solids, closer examination based on geometric computation reveals that they contain numerous voids and pockets. Most of them are of random nature, but some are binding sites providing surfaces to interact with other molecules.

Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.

In structural biology, the most critical issue is the availability of high-quality samples. "Structural-biology-grade" proteins must be generated in a quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance. The additional challenge for structural genomics is the need for high numbers of proteins at low cost where protein targets quite often have low sequence similarities, unknown properties and are poorly characterized.

Triclinic lysozyme at 0.65 A resolution.

The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 A resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.

Heparin-induced cis- and trans-dimerization modes of the thrombospondin-1 N-terminal domain.

Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by x-ray crystallography.

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain.

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds).

In situ proteolysis for protein crystallization and structure determination.

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.

Novel hexamerization motif is discovered in a conserved cytoplasmic protein from Salmonella typhimurium.

The cytoplasmic protein Stm3548 of unknown function obtained from a strain of Salmonella typhimurium was determined by X-ray crystallography at a resolution of 2.25 A. The asymmetric unit contains a hexamer of structurally identical monomers.

Crystal structure and putative function of small Toprim domain-containing protein from Bacillus stearothermophilus.

The crystal structure of the Midwest Center for Structural Genomics target APC35832, a 14.7-kDa cytosolic protein from Bacillus stearothermophilus, has been determined at 1.3 A resolution by the single anomalous diffraction method from a mercury soaked crystal.