Common Core Region Research Articles

A New ICEclc Subfamily Integrative and Conjugative Element Responsible for Horizontal Transfer of Biphenyl and Salicylic Acid Catabolic Pathway in the PCB-Degrading Strain Pseudomonas stutzeri KF716.

Integrative and conjugative elements (ICEs) are chromosomally integrated self-transmissible mobile genetic elements. Although some ICEs are known to carry genes for the degradation of aromatic compounds, information on their genetic features is limited. We identified a new member of the ICEclc family carrying biphenyl catabolic bph genes and salicylic acid catabolic sal genes from the PCB-degrading strain Pseudomonas stutzeri KF716. The 117-kb ICEbph-salKF716 contains common core regions exhibiting homology with those of degradative ICEclc from P. knackmussii B13 and ICEXTD from Azoarcus sp. CIB. A comparison of the gene loci collected from the public database revealed that several putative ICEs from P. putida B6-2, P, alcaliphila JAB1, P. stutzeri AN10, and P. stutzeri 2A20 had highly conserved core regions with those of ICEbph-salKF716, along with the variable region that encodes the catabolic genes for biphenyl, naphthalene, toluene, or phenol. These data indicate that this type of ICE subfamily is ubiquitously distributed within aromatic compound-degrading bacteria. ICEbph-salKF716 was transferred from P. stutzeri KF716 to P. aeruginosa PAO1 via a circular extrachromosomal intermediate form. In this study, we describe the structure and genetic features of ICEbph-salKF716 compared to other catabolic ICEs.

Common and distinct brain networks of autoscopic phenomena

ObjectiveAutoscopic phenomena (AP) are illusory own body reduplications characterized by the visual perception of a second own body in extrapersonal space, and include three main forms: autoscopic hallucination (AH), heautoscopy (HAS) and out-of-body-experience (OBE). Past research showed that lesions were heterogeneously distributed and affected many different brain regions within and across patients, while small case series suggested that AP lesions converge in temporo-parietal and parieto-occipital cortex. As only few studies investigated each form of AP separately, it remains unknown whether the three AP are characterized by common and distinct brain mechanisms. MethodsHere, we applied lesion network analysis in 26 neurological AP patients and determined their common and distinct functional connectivity patterns. ResultsWe report that all localize to a single common brain network at the bilateral temporo-parietal junction, further associated with specific patterns of functional connectivity, defining each type of AP. OBE resulted from a brain network connected to bilateral angular gyrus, right precuneus, and right inferior frontal gyrus, differing from AH with a brain network connected to bilateral precuneus, inferior temporal gyrus, and cerebellum. HAS resulted from a brain network connected to left inferior frontal gyrus, left insula and left parahippocampus. ConclusionThe present data identify the temporo-parietal junction as the common core region for AP and show that each form of AP recruits additional specific networks, associated with different sensorimotor and self-related sub-networks.

Tau Filament Self-Assembly and Structure: Tau as a Therapeutic Target.

Tau plays an important pathological role in a group of neurodegenerative diseases called tauopathies, including Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy and corticobasal degeneration. In each disease, tau self-assembles abnormally to form filaments that deposit in the brain. Tau is a natively unfolded protein that can adopt distinct structures in different pathological disorders. Cryo-electron microscopy has recently provided a series of structures for the core of the filaments purified from brain tissue from patients with different tauopathies and revealed that they share a common core region, while differing in their specific conformation. This structurally resolvable part of the core is contained within a proteolytically stable core region from the repeat domain initially isolated from AD tau filaments. Tau has recently become an important target for therapy. Recent work has suggested that the prevention of tau self-assembly may be effective in slowing the progression of Alzheimer's disease and other tauopathies. Here we review the work that explores the importance of tau filament structures and tau self-assembly mechanisms, as well as examining model systems that permit the exploration of the mode of action of potential inhibitors.

Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation.

The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen.IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.

Phenotype‐based clustering of glycosylation‐related genes by RNAi‐mediated gene silencing

Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, β1,4-galactosyltransferase I, β1,3-galactosyltransferase II, and β1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.

Harnessing apomictic reproduction in grasses: what we have learned from Paspalum

Apomixis is an alternative route of plant reproduction that produces individuals genetically identical to the mother plant through seeds. Apomixis is desirable in agriculture, because it guarantees the perpetuation of superior genotypes (i.e. heterotic hybrid seeds) by self-seeding without loss of hybrid vigour. The Paspalum genus, an archetypal model system for mining apomixis gene(s), is composed of about 370 species that have extremely diverse reproductive systems, including self-incompatibility, self-fertility, full sexual reproduction, and facultative or obligate apomixis. Barriers to interspecific hybridization are relaxed in this genus, allowing the production of new hybrids from many different parental combinations. Paspalum is also tolerant to various parental genome contributions to the endosperm, allowing analyses of how sexually reproducing crop species might escape from dosage effects in the endosperm. In this article, the available literature characterizing apomixis in Paspalum spp. and its use in breeding is critically reviewed. In particular, a comparison is made across species of the structure and function of the genomic region controlling apomixis in order to identify a common core region shared by all apomictic Paspalum species and where apomixis genes are likely to be localized. Candidate genes are discussed, either as possible genetic determinants (including homologs to signal transduction and RNA methylation genes) or as downstream factors (such as cell-to-cell signalling and auxin response genes) depending, respectively, on their co-segregation with apomixis or less. Strategies to validate the role of candidate genes in apomictic process are also discussed, with special emphasis on plant transformation in natural apomictic species.

Insights into the molecular evolution of oxytocin receptor ligand binding

The design and development of selective ligands for the human OT (oxytocin) and AVP (arginine vasopressin) receptors is a big challenge since the different receptor subtypes and their native peptide ligands display great similarity. Detailed understanding of the mechanism of OT's interaction with its receptor is important and may assist in the ligand- or structure-based design of selective and potent ligands. In the present article, we compared 69 OT- and OT-like receptor sequences with regards to their molecular evolution and diversity, utilized an in silico approach to map the common ligand interaction sites of recently published G-protein-coupled receptor structures to a model of the human OTR (OT receptor) and compared these interacting residues within a selection of different OTR sequences. Our analysis suggests the existence of a binding site for OT peptides within the common transmembrane core region of the receptor, but it appears extremely difficult to identify receptor or ligand residues that could explain the selectivity of OT to its receptors. We remain confident that the presented evolutionary overview and modelling approach will aid interpretation of forthcoming OTR crystal structures.

"Hot cores" in proteins: Comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms

BackgroundA wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures.ResultsThe construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions.ConclusionThis study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in the protein core and/or a better packing of alpha-helices with the rest of the structure, caused by the presence of hydrophobic beta-branched side chains.

An Essential Cell Cycle-regulated Nucleolar Protein Relocates to the Mitotic Spindle Where It Is Involved in Mitotic Progression in Trypanosoma brucei

TbNOP86 and TbNOP66 are two novel nucleolar proteins isolated in Trypanosoma brucei. They share 92.6% identity, except for an additional C-terminal domain of TbNOP86 of 182 amino acids in length. Both proteins are found in Trypanosomatidae, but similarity to other eukaryotic proteins could not be found. TbNOP86 and TbNOP66 are expressed at similar level in procyclic and bloodstream forms, although the relative level of expression of TbNOP66 is 11 times lower. TbNOP86 undergoes post-translational modifications, as it is found predominantly at 110 kDa compared with the predicted 86 kDa. Immunofluorescence of overexpressed ty-tagged TbNOP86 and TbNOP66 showed that both proteins accumulated in the nucleolus of G(1) cells. This was confirmed by the co-localization of an endogenous TbNOP86-myc with the nucleolar protein Nopp140. TbNOP86-ty localization is cell cycle-regulated, because it colocalizes with the mitotic spindle in mitotic cells. TbNOP86 is required for mitotic progression in both life stages as depleted cells are enriched in the G(2)/M phase. In procyclic cells, a reduced growth rate is accompanied by an accumulation of zoids (0N1K), 2N1K, and multinucleated cells (xNyK). The 2N1K cells are blocked in late mitosis as nucleolar segregation is completed. TbNOP86 depletion in bloodstream form caused a drastic growth inhibition producing cells bearing two kinetoplasts and an enlarged nucleus (1N(*)2K), followed by an accumulation of 2N2K cells with connected nuclei and xNyK cells. These studies of TbNOP86 provide a more comprehensive account of proteins involved in mitotic events in trypanosomes and should lead to the identification of partners with similar function.

Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon

NBU1 is a Bacteroides mobilizable transposon (MTn) that is integrated within the host chromosome and requires CTnDOT functions for its excision and transfer into a new host. The NBU1 integrase IntN1 has been classified as a tyrosine recombinase based on the presence of conserved residues. We created alanine mutants of the residues R291, K314, H393, R396, H419 and the conserved substitution Y429F and tested them for integration efficiency. The results suggest that these residues in IntN1 are important for integration, and Y429 could be the catalytic nucleophile. We employed suicide substrates and partially purified IntN1 to determine the positions of IntN1 cleavage within the 14 bp common core region that is identical in both NBU1 att sites. We show that IntN1 makes 7 bp staggered cuts on the top and bottom strands. From previous mutational analysis of the att sites, we show that two specific mutations near the site of bottom strand cleavage within this 7 bp region increased integration, and mutations of the two bases near top strand cleavage site had no effect on integration. These results indicate that IntN1 lacks the strict requirement for homology between the recombining sites seen with other tyrosine recombinases. We also show that phosphorothioate substitutions at the cleavage site and 1 bp downstream inhibited cleavage by IntN1. This differs from other studied tyrosine recombinases where inhibition occurs by substitutions at the cleavage site only.

NBU1 integrase: evidence for an altered recombination mechanism

NBU1 is a 10.3 kbp Bacteroides mobilizable transposon. A previous study had identified a 2.7 kbp segment of the excised circular intermediate that was sufficient to mediate integration of the element after transfer. This segment contained an integrase gene, intN1, and a region spanning the ends of the circular form within which integration occurred (attN1). The integrase protein, IntN1, appeared to be a member of the tyrosine recombinase family because it contains the canonical C-terminal RKHRHY [RK(H/K)R(H/W)Y] motif that characterizes members of that family. In this study, we describe an Escherichia coli-based integration assay system that has allowed us to characterize attN1 in detail. We first localized attN1 to a 250 bp region. We then used site-directed mutations to identify directly repeated sequences within attN1 that were required for site-specific integration. The locus of NBU1 site-specific integration in the Bacteroides thetaiotaomicron chromosome, attBT1-1, contains a 14 bp sequence that is identical to a 14 bp sequence that spans the joined ends of the NBU1 attN1 site (common core sequences). The effects of mutations in the common core were different from the expected results if NBU1 integration was similar to lambda integration. In particular single base changes near one end of the common core region, which introduced heterology, actually increased the frequency of integration. By contrast, compensating changes that restored homology in the common core region reduced the integration frequency. The recombination mechanism also differs from the one used by conjugative transposons that have coupling sequences between the sites of strand cleavage and exchange. These results indicate that although NBU1 integrase is considered to be a member of the tyrosine recombinase family, it catalyses an integrative recombination reaction that occurs by a different crossover mechanism.

Amino acid regions 572–579 and 657–666 of the spacer domain of ADAMTS13 provide a common antigenic core required for binding of antibodies in patients with acquired TTP

Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domain.

Amino Acid Regions 572–579 and 657–666 of the Spacer Domain of ADAMTS13 Provide a Common Antigenic Core Required for Binding of Antibodies in Patients with Acquired TTP.

Inhibitory antibodies against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura. Epitope-mapping studies revealed that antibodies binding to the cysteine-rich/spacer domains of ADAMTS13 are present in plasma of all patients analyzed so far. We have previously shown that a major antigenic determinant located within the spacer domain. To confirm these results we constructed a recombinant fragment consisting of the disintegrin/TSR1/cysteine-rich domains of ADAMTS13 and the spacer domain of ADAMTS1. The resulting ADAMTS13/ADAMTS1 chimera did not react with IgG present in plasma of a panel of patients with acquired TTP. To identify the amino acid residues that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of 15 hybrids (designated A–O) in which 5–10 amino acids of the ADAMTS13 spacer were exchanged for the homologous sequence of ADAMTS1. Plasma from 6 patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of residues 572–579 (hybrid C) and 657–666 (hybrid M) of ADAMTS13 for the corresponding sequence of ADAMTS1 completely abolished the binding of antibodies from all 6 patients. Other regions of the ADAMTS13 spacer were also involved in binding of antibodies from patient plasma. Regions 580–587 (D), 602–620 (G,H), 629–638 (J), and 667–767 (N) of the ADAMTS13 spacer domain contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). IgG derived from patient 1 required region 602–620 (G,H) for binding to the ADAMTS13 spacer (CGHM-epitope). For antibodies of patient 3, residues 564–571 (B), 580–587 (D), and 629–638 (J) were required (BCDJM-epitope), whereas replacement of residues 602–610 (G) and 629–638 (J) greatly diminished binding of antibodies derived from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of the patients, our results suggest that residues 572–579 (C) and 657–666 (M) comprise a common antigenic core region within the spacer domain that is crucial for binding of anti-ADAMTS13 antibodies in all six patients. Amino acid residues derived from other regions that spatially surround this common antigenic core further modulate binding of antibodies to the spacer domain.

Sequence and structural analysis of cellular retinoic acid-binding proteins reveals a network of conserved hydrophobic interactions.

Proteins in the intracellular lipid-binding protein (iLBP) family show remarkably high structural conservation despite their low-sequence identity. A multiple-sequence alignment using 52 sequences of iLBP family members revealed 15 fully conserved positions, with a disproportionately high number of these (n=7) located in the relatively small helical region. The conserved positions displayed high structural conservation based on comparisons of known iLBP crystal structures. It is striking that the beta-sheet domain had few conserved positions, despite its high structural conservation. This observation prompted us to analyze pair-wise interactions within the beta-sheet region to ask whether structural information was encoded in interacting amino acid pairs. We conducted this analysis on the iLBP family member, cellular retinoic acid-binding protein I (CRABP I), whose folding mechanism is under study in our laboratory. Indeed, an analysis based on a simple classification of hydrophobic and polar amino acids revealed a network of conserved interactions in CRABP I that cluster spatially, suggesting a possible nucleation site for folding. Significantly, a small number of residues participated in multiple conserved interactions, suggesting a key role for these sites in the structure and folding of CRABP I. The results presented here correlate well with available experimental evidence on folding of CRABPs and their family members and suggest future experiments. The analysis also shows the usefulness of considering pair-wise conservation based on a simple classification of amino acids, in analyzing sequences and structures to find common core regions among homologues.

Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs.

Arabidopsis ERF proteins such as DREB1, DREB2, and CBF1 bind to the dehydration-responsive element (DRE), which has the sequence TACCGACAT. Mutation analyses reveal that a central 5 bp CCGAC core of the DRE is the minimal sequence motif (designated as the DRE motif in this paper), to which the ERF domain fragment of CBF1 (CBF1-F) binds specifically with a binding K(d) at the nanomolar level. In contrast, the ERF domain fragment of the tobacco ERF2 (NtERF2-F) does not interact with the DRE motif, but restrictedly recognizes the sequence containing a minimal 6 bp GCCGCC motif (designated as the GCC motif in this paper). However, CBF1-F binds to the GCC motif with a binding activity similar to its binding activity for the DRE motif. These in vitro binding variations were further demonstrated through reporter cotransformation assays, suggesting that the DRE and GCC motifs are two similar sequence motifs sharing a common core region of CCGNC with a discriminating guanine base at the 5'-end of the GCC motif. Binding analyses with the mutated ERF domain show that such a unique binding of NtERF2-F to the GCC motif can be altered by the substitution of A14 with valine in beta-strand 2 of its ERF domain, the mutant NtERF2-F, ERFav, acquiring a binding to the DRE motif with a K(d) comparable to that for CBF1-F binding to the DRE motif. This demonstrates that A14 is an important determinant of the NtERF2-F binding specificity. A possible mechanism of the binding specificity determination is discussed.

Phylogenetic and structural analyses of the oxa1 family of protein translocases

Mitochondrial Oxa1p homologs have been shown to function in protein export and membrane insertion in bacteria, mitochondria and chloroplasts, but their mode of action, organismal distribution and evolutionary origins are poorly understood. All sequenced homologs of Oxa1p were retrieved from the databases and multiply aligned. All organisms with a fully sequenced genome possess at least one Oxa1p homolog showing that the family is truly ubiquitous. Most prokaryotes possess just one Oxa1p homolog, but several Gram-positive bacteria and one archaeon possess two, and eukaryotes may have as many as six. Although these proteins vary in length over a 5-fold range, they exhibit a common hydrophobic core region of about 200 residues. Multiple sequence alignments reveal conserved residues and provide the basis for structural and phylogenetic analyses that serve to characterize the Oxa1 family.

Phylogenetic and structural analyses of the oxa1 family of protein translocases

Mitochondrial Oxa1p homologs have been shown to function in protein export and membrane insertion in bacteria, mitochondria and chloroplasts, but their mode of action, organismal distribution and evolutionary origins are poorly understood. All sequenced homologs of Oxa1p were retrieved from the databases and multiply aligned. All organisms with a fully sequenced genome possess at least one Oxa1p homolog showing that the family is truly ubiquitous. Most prokaryotes possess just one Oxa1p homolog, but several Gram-positive bacteria and one archaeon possess two, and eukaryotes may have as many as six. Although these proteins vary in length over a 5-fold range, they exhibit a common hydrophobic core region of about 200 residues. Multiple sequence alignments reveal conserved residues and provide the basis for structural and phylogenetic analyses that serve to characterize the Oxa1 family.

Resolvase-like recombination performed by the TP901-1 integrase.

The site-specific recombination system of temperate lactococcal bacteriophage TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the lambda integrase family and second, in the presence of this integrase, a 56 bp attP fragment is sufficient for efficient recombination with the chromosomal attB site in the host Lactococcus lactis subsp. cremoris MG1363. In the present work, this attB site was analysed and a 43 bp attB region was found to be the smallest fragment able to participate fully in recombination. In vitro studies showed that the TP901-1 integrase binds this 43 bp attB fragment, the 56 bp attP and a larger attP fragment with equal affinity. Mutational analysis of the 5 bp common core region (TCAAT) showed that the TC dinucleotide is essential for recombination, but not for binding of the integrase, whereas none of the last three bases are important for recombination. When a number of attL sites, obtained by recombination between an attB site containing a mutation in this TC dinucleotide and a wild-type attP site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These results are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for attB sites with a decreased ability to participate in recombination, two bases located outside the core region of attB were shown to be involved in the in vitro binding of the TP901-1 integrase.

The Site-Specific Recombination System of theLactobacillusSpecies Bacteriophage A2 Integrates in Gram-Positive and Gram-Negative Bacteria

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA ofLactobacillusspp. has been identified. Twoorfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). Theorfadjacent toattPpredicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The otherorfencodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region withattP. TheattBsite is located at the 3′ end of the transfer RNALeugene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such asLactococcus lactisand even intoEscherichia coli. InLc. lactis, integration occurs in a previously unknown intergenic region, whereas inE. coli, it maps within therrnDoperon, 5′ ofrrsDgene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in theattBsequence, sinceLc. lactisandE. colionly share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.

Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding.

Uracil phosphoribosyltransferase (UPRTase) catalyzes the transfer of a ribosyl phosphate group from alpha-D-5-phosphoribosyl-1-pyrophosphate to the N1 nitrogen of uracil. The UPRTase from the opportunistic pathogen Toxoplasma gondii is a rational target for antiparasitic drug design. To aid in structure-based drug design studies against toxoplasmosis, the crystal structures of the T.gondii apo UPRTase (1.93 A resolution), the UPRTase bound to its substrate, uracil (2.2 A resolution), its product, UMP (2.5 A resolution), and the prodrug, 5-fluorouracil (2.3 A resolution), have been determined. These structures reveal that UPRTase recognizes uracil through polypeptide backbone hydrogen bonds to the uracil exocyclic O2 and endocyclic N3 atoms and a backbone-water-exocyclic O4 oxygen hydrogen bond. This stereochemical arrangement and the architecture of the uracil-binding pocket reveal why cytosine and pyrimidines with exocyclic substituents at ring position 5 larger than fluorine, including thymine, cannot bind to the enzyme. Strikingly, the T. gondii UPRTase contains a 22 residue insertion within the conserved PRTase fold that forms an extended antiparallel beta-arm. Leu92, at the tip of this arm, functions to cap the active site of its dimer mate, thereby inhibiting the escape of the substrate-binding water molecule.