A Distinct Type of Pilus from the Human Microbiome.

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key ß sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal ß strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

Structure and function of the DUF2233 domain in bacteria and in the human mannose 6-phosphate uncovering enzyme.

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase ("uncovering enzyme" (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.

Crystal structure of a member of a novel family of dioxygenases (PF10014) reveals a conserved cupin fold and active site.

PF10014 is a novel family of 2-oxyglutarate-Fe(2+) -dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the ß-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site.

Crystal structure of a putative quorum sensing-regulated protein (PA3611) from the Pseudomonas-specific DUF4146 family.

Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family.

Structural analysis of arabinose-5-phosphate isomerase from Bacteroides fragilis and functional implications.

The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7 Šresolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.

Structure and function of a novel LD-carboxypeptidase a involved in peptidoglycan recycling.

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-Šresolution. 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.

Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization.

The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg(2+) , was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down 'active site'. Homology modelling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.

The structure of Mlc titration factor A (MtfA/YeeI) reveals a prototypical zinc metallopeptidase related to anthrax lethal factor.

MtfA of Escherichia coli (formerly YeeI) was previously identified as a regulator of the phosphoenolpyruvate (PEP)-dependent:glucose phosphotransferase system. MtfA homolog proteins are highly conserved, especially among beta- and gammaproteobacteria. We determined the crystal structures of the full-length MtfA apoenzyme from Klebsiella pneumoniae and its complex with zinc (holoenzyme) at 2.2 and 1.95 Å, respectively. MtfA contains a conserved H(149)E(150)XXH(153)+E(212)+Y(205) metallopeptidase motif. The presence of zinc in the active site induces significant conformational changes in the region around Tyr205 compared to the conformation of the apoenzyme. Additionally, the zinc-bound MtfA structure is in a self-inhibitory conformation where a region that was disordered in the unliganded structure is now observed in the active site and a nonproductive state of the enzyme is formed. MtfA is related to the catalytic domain of the anthrax lethal factor and the Mop protein involved in the virulence of Vibrio cholerae, with conservation in both overall structure and in the residues around the active site. These results clearly provide support for MtfA as a prototypical zinc metallopeptidase (gluzincin clan).

Structural basis of murein peptide specificity of a gamma-D-glutamyl-l-diamino acid endopeptidase.

The crystal structures of two homologous endopeptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme were determined at 1.05 and 1.60 A resolution, respectively, and contain a bacterial SH3-like domain (SH3b) and a ubiquitous cell-wall-associated NlpC/P60 (or CHAP) cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that these two proteins act as gamma-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.

Structural and functional characterizations of SsgB, a conserved activator of developmental cell division in morphologically complex actinomycetes.

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 A resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic "whirly" single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.

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.

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.

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.

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).

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 Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01 Šusing 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.

The structure of the first representative of Pfam family PF09836 reveals a two-domain organization and suggests involvement 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.

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.

The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Šby 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.

The structure of Jann_2411 (DUF1470) from Jannaschia sp. at 1.45†Šresolution reveals a new fold (the ABATE domain) and suggests its possible role as a transcription regulator.

The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45 Šby multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.

Structure of LP2179, the first representative of Pfam family PF08866, suggests a new fold with a role in amino-acid metabolism.

The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+ß fold comprising two ß-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.

The structure of the first representative of Pfam family PF06475 reveals a new fold with possible involvement in glycolipid metabolism.

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 ß-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.

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 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.

Structure of an essential bacterial protein YeaZ (TM0874) from Thermotoga maritima at 2.5†Šresolution.

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 Šresolution. 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.