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1.  Structure and Function of a Novel ld-Carboxypeptidase A Involved in Peptidoglycan Recycling 
Journal of Bacteriology  2013;195(24):5555-5566.
Approximately 50% of cell wall peptidoglycan in Gram-negative bacteria is recycled with each generation. The primary substrates used for peptidoglycan biosynthesis and recycling in the cytoplasm are GlcNAc-MurNAc(anhydro)-tetrapeptide and its degradation product, the free tetrapeptide. This complex process involves ∼15 proteins, among which the cytoplasmic enzyme ld-carboxypeptidase A (LdcA) catabolizes the bond between the last two l- and d-amino acid residues in the tetrapeptide to form the tripeptide, which is then utilized as a substrate by murein peptide ligase (Mpl). LdcA has been proposed as an antibacterial target. The crystal structure of Novosphingobium aromaticivorans DSM 12444 LdcA (NaLdcA) was determined at 1.89-Å resolution. The enzyme was biochemically characterized and its interactions with the substrate modeled, identifying residues potentially involved in substrate binding. Unaccounted electron density at the dimer interface in the crystal suggested a potential site for disrupting protein-protein interactions should a dimer be required to perform its function in bacteria. Our analysis extends the identification of functional residues to several other homologs, which include enzymes from bacteria that are involved in hydrocarbon degradation and destruction of coral reefs. The NaLdcA crystal structure provides an alternate system for investigating the structure-function relationships of LdcA and increases the structural coverage of the protagonists in bacterial cell wall recycling.
doi:10.1128/JB.00900-13
PMCID: PMC3889619  PMID: 24123814
2.  Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum  
DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols.
Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
doi:10.1107/S090744491104978X
PMCID: PMC3322598  PMID: 22505259
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
3.  The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu 
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.
doi:10.1002/pro.497
PMCID: PMC3005784  PMID: 20836087
Neisseria gonorrhoeae; NGO1391; UniProt Q5F6Z8; Pfam PF05076; suppressor of fused; sufu-like; structural genomics
4.  Structural and Sequence Analysis of Imelysin-Like Proteins Implicated in Bacterial Iron Uptake 
PLoS ONE  2011;6(7):e21875.
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.
doi:10.1371/journal.pone.0021875
PMCID: PMC3143127  PMID: 21799754
5.  Crystal Structure of the First Eubacterial Mre11 Nuclease Reveals Novel Features that may Discriminate Substrates During DNA Repair 
Journal of molecular biology  2010;397(3):647-663.
Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks (DSBs). As x-ray structural information has only been available for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is the Mre11 endo/exonuclease from T. maritima (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only ∼20%. However, they differ substantially in their DNA specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA specificity domain are not. The structural differences likely affect how Mre11s from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on ssDNA and dsDNA substrates, respectively.
doi:10.1016/j.jmb.2010.01.049
PMCID: PMC2839085  PMID: 20122942
Mre11; SbcD; endonuclease; exonuclease; crystal structure; DNA repair; structural genomics; Thermotoga maritima
6.  Structure of a tryptophanyl-tRNA synthetase containing an iron–sulfur cluster 
The crystal structure of tryptophanyl-tRNA synthetase from T. maritima unexpectedly revealed an iron–sulfur cluster bound to the tRNA anticodon-binding region.
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 6.1.1.2) 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-x 22-C-x 6-C-x 2-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.
doi:10.1107/S1744309110037619
PMCID: PMC2954223  PMID: 20944229
TM0492; tryptophanyl-tRNA ligase; tryptophanyl-tRNA synthetase class I; iron–sulfur clusters; structural genomics
7.  Structure of BT_3984, a member of the SusD/RagB family of nutrient-binding molecules 
The crystal structure of BT_3984, a SusD-family protein, reveals a TPR N-terminal region providing support for a loop-rich C-terminal subdomain and suggests possible interfaces involved in sus complex formation.
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.
doi:10.1107/S1744309110032999
PMCID: PMC2954216  PMID: 20944222
structural genomics; starch-utilization system; gut microbiome; metagenomics
8.  The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu 
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.
doi:10.1002/pro.497
PMCID: PMC3005784  PMID: 20836087
Neisseria gonorrhoeae; NGO1391; UniProt Q5F6Z8; Pfam PF05076; suppressor of fused; sufu-like; structural genomics
9.  Structure of a putative NTP pyrophosphohydrolase: YP_001813558.1 from Exiguobacterium sibiricum 255-15 
The crystal structure of a putative NTP pyrophosphohydrolase, YP_001813558.1 from E. sibiricum, reveals a novel segment-swapped linked-dimer assembly.
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.
doi:10.1107/S1744309110025534
PMCID: PMC2954211  PMID: 20944217
structural genomics; putative NTP pyrophosphohydrolase; MazG nucleotide pyrophosphohydrolase; dUTPases
10.  Structure of Bacteroides thetaiotaomicron BT2081 at 2.05 Å resolution: the first structural representative of a new protein family that may play a role in carbohydrate metabolism 
The crystal structure of BT2081 from B. thetaiotaomicron reveals a two-domain protein with a putative carbohydrate-binding site in the C-­terminal domain.
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.
doi:10.1107/S1744309110028228
PMCID: PMC2954218  PMID: 20944224
gut microbiome; sugars; structural genomics; immunoglobulin-like fold; jelly-roll fold
11.  Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger 
The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing.
Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2_DESHY from the anaerobic dehalogenating bacterium Desulfito­bacterium hafniense DCB-2, Q2LQ23_SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63_THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63_THEAC and Q2LQ23_SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2_DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63_THEAC and Q2LQ23_SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63_THEAC and Q2LQ23_SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63_THEAC, but is absent from the NTD of Q2LQ23_SYNAS. Second, whereas the structure of the CTD of Q2LQ23_SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure of Q9HJ63_THEAC is atypical for a treble-cleft zinc finger in that three Cys side chains and an Asp side chain are within coordination distance of the zinc.
doi:10.1107/S1744309110020166
PMCID: PMC2954224  PMID: 20944230
Pfam family PF02663; metalloproteins; domain swapping; structural genomics; methanogenesis
12.  Structure of a membrane-attack complex/perforin (MACPF) family protein from the human gut symbiont Bacteroides thetaiotaomicron  
The crystal structure of a novel MACPF protein, which may play a role in the adaptation of commensal bacteria to host environments in the human gut, was determined and analyzed.
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.
doi:10.1107/S1744309110023055
PMCID: PMC2954219  PMID: 20944225
MACPF; membrane-attack complexes; perforins; transmembrane pores; pathogenesis
13.  The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes 
The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes.
Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)+-dependent oxidative decarboxyl­ation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD+ has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP+ and NAD+. The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between β5 and β6) and Arg365′ (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.
doi:10.1107/S1744309110021688
PMCID: PMC2954222  PMID: 20944228
tyrosine biosynthesis; prephenate; chorismate; Haemophilus influenzae; structural genomics
14.  Structure of the γ-d-glutamyl-l-diamino acid endopeptidase YkfC from Bacillus cereus in complex with l-Ala-γ-d-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases 
The crystal structure of the highly specific γ-d-glutamyl-l-diamino acid endopeptidase YkfC from Bacillus cereus in complex with l-Ala-γ-d-Glu reveals the structural basis for the substrate specificity of NlpC/P60-family cysteine peptidases.
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.
doi:10.1107/S1744309110021214
PMCID: PMC2954226  PMID: 20944232
γ-d-glutamyl-l-diamino acid endopeptidase; cell-wall recycling; NlpC/P60; SH3b; cysteine peptidases; enzyme specificity
15.  Crystal Structure of Histidine Phosphotransfer Protein ShpA, an Essential Regulator of Stalk Biogenesis in Caulobacter crescentus 
Journal of molecular biology  2009;390(4):686-698.
Cell cycle regulated stalk biogenesis in Caulobacter crescentus is controlled by a multi-step phosphorelay system consisting of the hybrid histidine kinase ShkA, the histidine-phosphotransfer protein ShpA and the response regulator TacA. ShpA shuttles phosphoryl groups between ShkA and TacA. When phosphorylated, TacA triggers a downstream transcription cascade for stalk synthesis in an RpoN-dependent manner. The crystal structure of ShpA was determined to 1.52 Å resolution. ShpA belongs to a family of monomeric histidine phosphotransfer (HPt) proteins, which feature a highly conserved four-helix bundle. The phosphorylatable histidine, His56, is located on the surface of the helix bundle and is fully solvent exposed. One end of the four-helix bundle in ShpA is shorter compared to other characterized histidine phosphotransfer proteins, whereas the face that potentially interacts with the response regulators is structurally conserved. Similarities of the interaction surface around the phosphorylation site suggest that ShpA is likely to share a common mechanism for molecular recognition and phosphotransfer with yeast phosphotransfer protein YPD1 despite low overall sequence similarity.
doi:10.1016/j.jmb.2009.05.023
PMCID: PMC2726009  PMID: 19450606
Stalk biogenesis; phosphorelay; two-component signal transduction; Histidine phosphotransfer protein (HPt)
16.  Structure of the first representative of Pfam family PF04016 (DUF364) reveals enolase and Rossmann-like folds that combine to form a unique active site with a possible role in heavy-metal chelation 
The crystal structure of the first representative of DUF364 family reveals a combination of enolase N-terminal-like and C-terminal Rossmann-like folds. Analysis of the interdomain cleft combined with sequence and genome context conservation among homologs, suggests a unique catalytic site likely involved in the synthesis of a flavin or pterin derivative.
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.
doi:10.1107/S1744309110007517
PMCID: PMC2954201  PMID: 20944207
structural genomics; domains of unknown function; rare metals; siderophores; pterins
17.  A conserved fold for fimbrial components revealed by the crystal structure of a putative fimbrial assembly protein (BT1062) from Bacteroides thetaiotaomicron at 2.2 Å resolution 
The crystal structure of BT1062 from Bacteroides thetaiotaomicron revealed a conserved fold that is widely adopted by fimbrial components.
BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.
doi:10.1107/S1744309110006548
PMCID: PMC2954217  PMID: 20944223
DUF1812; PF08842; pili; fimbriae; BT1062; Mfa2; PGN0288; PG0179
18.  The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role 
The crystal structure of SSO2064, the first structural representative of Pfam family PF01796 (DUF35), reveals a two-domain architecture comprising an N-terminal zinc-ribbon domain and a C-terminal OB-fold domain. Analysis of the domain architecture, operon organization and bacterial orthologs combined with the structural features of SSO2064 suggests a role involving acyl-CoA binding for this family of proteins.
SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).
doi:10.1107/S1744309110002514
PMCID: PMC2954200  PMID: 20944206
structural genomics; domains of unknown function; acyl-carrier proteins; acyl-coA; polyketide biosynthesis
19.  Structures of the first representatives of Pfam family PF06684 (DUF1185) reveal a novel variant of the Bacillus chorismate mutase fold and suggest a role in amino-acid metabolism 
Structures of the first representatives of PF06684 (DUF1185) reveal a Bacillus chorismate mutase-like fold with a potential role in amino-acid synthesis.
The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.
doi:10.1107/S1744309109050647
PMCID: PMC2954203  PMID: 20944209
domain of unknown function; structural genomics; chorismate mutase; amino acids; pH-dependent; salt-dependent
20.  Structures of the first representatives of Pfam family PF06938 (DUF1285) reveal a new fold with repeated structural motifs and possible involvement in signal transduction 
The crystal structures of SPO0140 and Sbal_2486 revealed a two-domain structure that adopts a novel fold. Analysis of the interdomain cleft suggests a nucleotide-based ligand with a genome context indicating signaling as a possible role for this family.
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.
doi:10.1107/S1744309109050416
PMCID: PMC2954208  PMID: 20944214
structural genomics; domain of unknown function; domain duplication; signaling; oxidative stress
21.  The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function 
The crystal structure of the BVU2987 gene product from B. vulgatus (UniProt A6L4L1) reveals that members of the new Pfam family PF11396 (domain of unknown function; DUF2874) are similar to β-lactamase inhibitor protein and YpmB.
Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.
doi:10.1107/S1744309109046788
PMCID: PMC2954215  PMID: 20944221
BVU2987; DUF2874; PF11396; human gut microbiome; β-lactamase inhibitor protein-like fold; putative inhibitor proteins
22.  Bacterial Pleckstrin Homology Domains: A Prokaryotic Origin for the PH Domain 
Journal of Molecular Biology  2010;396(1):31-46.
Pleckstrin homology (PH) domains have been identified only in eukaryotic proteins to date. We have determined crystal structures for three members of an uncharacterized protein family (Pfam PF08000), which provide compelling evidence for the existence of PH-like domains in bacteria (PHb). The first two structures contain a single PHb domain that forms a dome-shaped, oligomeric ring with C5 symmetry. The third structure has an additional helical hairpin attached at the C-terminus and forms a similar but much larger ring with C12 symmetry. Thus, both molecular assemblies exhibit rare, higher-order, cyclic symmetry but preserve a similar arrangement of their PHb domains, which gives rise to a conserved hydrophilic surface at the intersection of the β-strands of adjacent protomers that likely mediates protein–protein interactions. As a result of these structures, additional families of PHb domains were identified, suggesting that PH domains are much more widespread than originally anticipated. Thus, rather than being a eukaryotic innovation, the PH domain superfamily appears to have existed before prokaryotes and eukaryotes diverged.
doi:10.1016/j.jmb.2009.11.006
PMCID: PMC2817789  PMID: 19913036
PH, Pleckstrin homology; PHb, bacterial PH domain; PTB, phosphotyrosine binding; VPS36, vacuolar protein sorting protein 36; DUF1696, domain of unknown function family 1696; JCSG, Joint Center for Structural Genomics; MAD, multiwavelength anomalous diffraction; PEG, polyethylene glycol; asu, asymmetric unit; PDB, Protein Data Bank; PIPE, Polymerase Incomplete Primer Extension; TEV, tobacco etch virus; TCEP, tris(2-carboxyethyl)phosphine–HCl; SSRL, Stanford Synchrotron Radiation Lightsource; ALS, Advanced Light Source; Pleckstrin homology (PH) domain; bacterial PH domain (PHb); higher-order symmetry; protein assembly; protein evolution
23.  Structure of the first representative of Pfam family PF09410 (DUF2006) reveals a structural signature of the calycin superfamily that suggests a role in lipid metabolism 
NE1406, the first structural representative of PF09410, reveals a lipocalin-like fold with features that suggest involvement in lipid metabolism. In addition, NE1406 provides potential structural templates for two other protein families (PF07143 and PF08622).
The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-­terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively.
doi:10.1107/S1744309109037749
PMCID: PMC2954199  PMID: 20944205
structural genomics; domains of unknown function; calycin; lipocalin; fatty-acid binding proteins
24.  Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding 
The crystal structures of two orthologous proteins from different Shewanella species have uncovered a resemblance to CRAL-TRIO carrier proteins, which suggest that they function as transporters of small nonpolar molecules. One protein adopts an open conformation, while the other adopts a closed structure that may act as a conformational switch in the transport of ligands at the membrane surface.
The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25 Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/β SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the ‘open’ conformation (YP_001095227.1), these helices are 15 Å apart, leading to the creation of a deep nonpolar cavity. In the ‘closed’ structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their ‘open’ monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.
doi:10.1107/S1744309109042481
PMCID: PMC2954212  PMID: 20944218
YP_001095227.1; YP_749275.1; SpoIIAA-like proteins
25.  The structure of the first representative of Pfam family PF09836 reveals a two-domain organization and suggests involvement in transcriptional regulation 
The crystal structure of the NGO1945 gene product from N. gonorrhoeae (UniProt Q5F5IO) reveals that the N-terminal domain assigned as a domain of unknown function (DUF2063) is likely to bind DNA and that the protein may be involved in transcriptional regulation.
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.
doi:10.1107/S1744309109022672
PMCID: PMC2954202  PMID: 20944208
NGO1945; PF09836; DUF2063; putative DNA-binding proteins; putative transcription regulators; structural genomics

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