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author:("grichnik, A.")
1.  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
2.  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)
3.  Crystal structure of a novel Sm-like protein of putative cyanophage origin at 2.60 Å resolution 
Proteins  2009;75(2):296-307.
ECX21941 represents a very large family (over 600 members) of novel, ocean metagenome–specific proteins identified by clustering of the dataset from the Global Ocean Sampling expedition. The crystal structure of ECX21941 reveals unexpected similarity to Sm/LSm proteins, which are important RNA-binding proteins, despite no detectable sequence similarity. The ECX21941 protein assembles as a homopentamer in solution and in the crystal structure when expressed in Escherichia coli and represents the first pentameric structure for this Sm/LSm family of proteins, although the actual oligomeric form in vivo is currently not known. The genomic neighborhood analysis of ECX21941 and its homologs combined with sequence similarity searches suggest a cyanophage origin for this protein. The specific functions of members of this family are unknown, but our structure analysis of ECX21941 indicates nucleic acid-binding capabilities and suggests a role in RNA and/or DNA processing.
doi:10.1002/prot.22360
PMCID: PMC2785455  PMID: 19173316
Structural genomics; metagenomics; nucleic acid binding; Sm-like; viral protein
5.  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
6.  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
7.  The structure of Jann_2411 (DUF1470) from Jannaschia sp. at 1.45 Å resolution reveals a new fold (the ABATE domain) and suggests its possible role as a transcription regulator 
The crystal structure of the first representative of the Pfam PF07336 (DUF1470) family reveals a two-domain organization that contains a new fold, termed the ABATE domain, at the N-terminus and a treble-clef zinc finger that is likely to bind DNA at the C-terminus.
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 Å by 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.
doi:10.1107/S1744309109025196
PMCID: PMC2954205  PMID: 20944211
structural genomics; environmental stress; domains of unknown function; Pfam; bound metal identification
8.  RNA Polymerase I Stability Couples Cellular Growth to Metal Availability 
Molecular cell  2013;51(1):105-115.
Summary
Zinc is an essential cofactor of all major eukaryotic RNA polymerases. How the activity of these enzymes is coordinated or regulated according to cellular zinc levels is largely unknown. Here we show that the stability of RNA Polymerase I (RNAPI) is tightly coupled to zinc availability in vivo. In zinc deficiency, RNAPI is specifically degraded by proteolysis in the vacuole in a pathway dependent on the exportin Xpo1p and deubiquitination of the RNAPI large subunit Rpa190p by Ubp2p and Ubp4p. RNAPII is unaffected, which allows for expression of genes required in zinc deficiency. RNAPI export to the vacuole is required for survival during zinc starvation, suggesting that degradation of zinc-binding subunits might provide a last resort zinc reservoir. These results reveal a hierarchy of cellular transcriptional activities during zinc starvation, and show that degradation of the most active cellular transcriptional machinery couples cellular growth and proliferation to zinc availability.
doi:10.1016/j.molcel.2013.05.005
PMCID: PMC3713077  PMID: 23747013
9.  Terminate and make a loop: regulation of transcriptional directionality 
Trends in Biochemical Sciences  2014;39(7):319-327.
Highlights
•Transcriptional directionality is controlled by premature transcription termination.•Transcriptional directionality is enforced by gene looping.•mRNA-specific termination signals and factors are required for gene looping.
Bidirectional promoters are a common feature of many eukaryotic organisms from yeast to humans. RNA Polymerase II that is recruited to this type of promoter can start transcribing in either direction using alternative DNA strands as the template. Such promiscuous transcription can lead to the synthesis of unwanted transcripts that may have negative effects on gene expression. Recent studies have identified transcription termination and gene looping as critical players in the enforcement of promoter directionality. Interestingly, both mechanisms share key components. Here, we focus on recent findings relating to the transcriptional output of bidirectional promoters.
doi:10.1016/j.tibs.2014.05.001
PMCID: PMC4085477  PMID: 24928762
bidirectional promoters; transcriptional termination; gene loops
10.  Twinned caesium cerium(IV) penta­fluoride 
Single-crystals of CsCeF5 were synthesized hydro­thermally. The crystal under investigation was twinned by pseudo-merohedry with a twofold rotation around the c axis as an additional twinning operation. The crystal structure is built of layers of distorted edge- and corner-sharing CeF8 square-anti­prisms. The Cs+ cations are located between the layers and exhibit coordination numbers of nine. Upon compression, CsCeF5 undergoes an irreversible phase transition at about 1 GPa.
doi:10.1107/S1600536814003286
PMCID: PMC3998492  PMID: 24764931
11.  Structure of a Novel Winged-Helix Like Domain from Human NFRKB Protein 
PLoS ONE  2012;7(9):e43761.
The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370–495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.
doi:10.1371/journal.pone.0043761
PMCID: PMC3439487  PMID: 22984442
12.  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
13.  Crystal structure and stability of Tl2CO3 at high pressures 
The crystal structure of dithallium carbonate, Tl2CO3 (C2/m, Z = 4), is stable to 5.82 GPa and does not undergo any phase transitions at lower pressures as reported previously. At higher pressures, the material undergoes a phase transition that destroys the single crystal.
The crystal structure of dithallium carbonate, Tl2CO3 (C2/m, Z = 4), was investigated at pressures of up to 7.4 GPa using single-crystal X-ray diffraction in a diamond anvil cell. It is stable to at least 5.82 GPa. All atoms except for one of the O atoms lie on crystallographic mirror planes. At higher pressures, the material undergoes a phase transition that destroys the single crystal.
doi:10.1107/S0108270110005652
PMCID: PMC2855587  PMID: 20203393
14.  Yeast Sen1 Helicase Protects the Genome from Transcription-Associated Instability 
Molecular Cell  2011;41(1):21-32.
Summary
Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
Graphical Abstract
Highlights
► Nascent RNA forms hybrids with underwound DNA upstream of elongating Pol II ► Single-stranded DNA so formed is prone to damage which results in genome instability ► Sen1 helicase acts to remove R loops by resolving RNA:DNA hybrids ► Sen1 function in Pol II elongation and termination may relate to R loop resolution
doi:10.1016/j.molcel.2010.12.007
PMCID: PMC3314950  PMID: 21211720
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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