[Show abstract][Hide abstract] ABSTRACT: Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution), have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function.
PLoS ONE 01/2011; 6(7):e21875. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, "closed" conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.
PLoS ONE 01/2011; 6(7):e22013. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.
Journal of Biological Chemistry 01/2011; 286(14):12450-60. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.
Journal of Molecular Biology 12/2010; 404(3):403-17. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam.
Protein Science 11/2010; 19(11):2131-40. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1137-42. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40-99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1174-81. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1218-25. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1254-60. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7 Å and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1274-80. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel aminoacyl-tRNA synthetase that contains an iron-sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 188.8.131.52) reveals an iron-sulfur [4Fe-4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an L-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe-4S] cluster-binding motif (C-x₂₂-C-x₆-C-x₂-C). It is speculated that the iron-sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1326-34. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6 Å resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006), Proteins, 65, 1046-1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1347-53. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1354-64. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1237-44. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05 Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a β-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a β-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal β-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1287-96. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Membrane-attack complex/perforin (MACPF) proteins are transmembrane pore-forming proteins that are important in both human immunity and the virulence of pathogens. Bacterial MACPFs are found in diverse bacterial species, including most human gut-associated Bacteroides species. The crystal structure of a bacterial MACPF-domain-containing protein BT_3439 (Bth-MACPF) from B. thetaiotaomicron, a predominant member of the mammalian intestinal microbiota, has been determined. Bth-MACPF contains a membrane-attack complex/perforin (MACPF) domain and two novel C-terminal domains that resemble ribonuclease H and interleukin 8, respectively. The entire protein adopts a flat crescent shape, characteristic of other MACPF proteins, that may be important for oligomerization. This Bth-MACPF structure provides new features and insights not observed in two previous MACPF structures. Genomic context analysis infers that Bth-MACPF may be involved in a novel protein-transport or nutrient-uptake system, suggesting an important role for these MACPF proteins, which were likely to have been inherited from eukaryotes via horizontal gene transfer, in the adaptation of commensal bacteria to the host environment.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1297-305. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1167-73. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+β fold comprising two β-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1205-10. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of PA1994 from Pseudomonas aeruginosa, a member of the Pfam PF06475 family classified as a domain of unknown function (DUF1089), reveals a novel fold comprising a 15-stranded β-sheet wrapped around a single α-helix that assembles into a tight dimeric arrangement. The remote structural similarity to lipoprotein localization factors, in addition to the presence of an acidic pocket that is conserved in DUF1089 homologs, phospholipid-binding and sugar-binding proteins, indicate a role for PA1994 and the DUF1089 family in glycolipid metabolism. Genome-context analysis lends further support to the involvement of this family of proteins in glycolipid metabolism and indicates possible activation of DUF1089 homologs under conditions of bacterial cell-wall stress or host-pathogen interactions.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1211-7. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: YeaZ is involved in a protein network that is essential for bacteria. The crystal structure of YeaZ from Thermotoga maritima was determined to 2.5 Å resolution. Although this protein belongs to a family of ancient actin-like ATPases, it appears that it has lost the ability to bind ATP since it lacks some key structural features that are important for interaction with ATP. A conserved surface was identified, supporting its role in the formation of protein complexes.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2010; 66(Pt 10):1230-6. · 0.55 Impact Factor