DNA Binding Proteins of the Filamentous Phages CTXφ and VGJφ of Vibrio cholerae

The native product of open reading frame 112 (orf112) and a recombinant variant of the RstB protein, encoded by Vibrio cholerae pathogen-specific bacteriophages VGJφ and CTXφ, respectively, were purified to more than 90% homogeneity. Orf112 protein was shown to specifically bind single-stranded genomic DNA of VGJφ; however, RstB protein unexpectedly bound double-stranded DNA in addition to the single-stranded genomic DNA. The DNA binding properties of these proteins may explain their requirement for the rolling circle replication of the respective phages and RstB''s requirement for single-stranded-DNA chromosomal integration of CTXφ phage dependent on XerCD recombinases.Vibrio cholerae, the etiologic agent of cholera, is a gram-negative bacterium which hosts several specific filamentous phages (1, 7, 8, 9, 10, 11, 13). CTXφ phage has been the most studied due to its role in pathogenicity and horizontal gene transfer (6). This phage is usually integrated into the genomes of toxigenic strains of V. cholerae, but it is also able to replicate directly from the bacterial chromosome (6) and to produce infective phage particles with potential for transducing the cholera toxin genes into nonpathogenic environmental strains (6, 13). Another filamentous phage important for its role in horizontal gene transfer is VGJφ, which is able to recombine with the CTXφ genome to originate a hybrid phage endowed with the full potential for virulence conversion. The hybrid phage shows an increased infectivity due to its specificity for the receptor mannose-sensitive hemagglutinin (receptor mannose-sensitive hemagglutinin pilus), which is ubiquitous among environmental strains (1, 2). Therefore, elucidating the biology of these phages is crucial for understanding the evolution of bacterial pathogenesis.The genomes of CTXφ and VGJφ carry the putative homologous rstB and open reading frame 112 (orf112) genes, respectively. The requirement of rstB for the integration of CTXφ into the bacterial chromosome has been described (14). However, the biochemical function of the gene product has not been elucidated. Genes rstB and orf112 are positional and size homologues of genes encoding single-stranded DNA (ssDNA)-binding proteins (SSB) in other filamentous phages (1). It is expected that the proteins encoded by rstB and orf112 exert similar functions in the biology of their respective phages (1). Thus, we wanted to evaluate the ssDNA-binding activity of these ORF products.To asses whether the Orf112 product and RstB have SSB activity, sufficient amounts of pure proteins are required. This paper describes quick purification protocols used to obtain both protein species and the evaluation of their DNA binding activities. The Orf112 protein was obtained from V. cholerae strain 569B (serogroup O1, Inaba classical biotype) infected with VGJφ, which expresses high levels of the protein. The infected bacteria were inoculated into 300 ml of LB broth and were cultured with shaking overnight at 200 rpm and 37°C. Parallel uninfected batches of 569B were also processed. Cells were collected by centrifugation for 15 min at 9,000 × g and at 4°C and stored at −20°C until processed.A recombinant rstB gene with a hexahistidine tag coding region fused to the C terminus of the respective protein product (RstB-His) was constructed by cloning the gene into the expression vector pBAD/Myc-HisC (Invitrogen). The rstB gene was PCR amplified from the CTXφ genome using the oligonucleotides CNC06-171 (5′-AGTTCCATGGGGAAATTATGGGTGATAAT-3′) and CNC06-173 (5′-CATCAAGCTTTAATGGGT-3′), which introduce restriction sites for NcoI and HindIII at the amplicon ends. The amplified fragment was digested with both enzymes and cloned into the same sites of pBAD/Myc-HisC. In the resultant construction, named pBAD/Myc-HisC-rstB 9, expression of the recombinant protein is inducible by arabinose.Plasmid pBAD/Myc-HisC-rstB 9 was electroporated into Escherichia coli Top 10. A 1-ml sample of an overnight, 5-ml, ampicillin-supplemented LB broth culture of transformed Top 10 was inoculated into 300 ml of fresh broth. The culture was incubated with orbital shaking at 200 rpm and 37°C until it reached an optical density at 600 nm of 0.5. To induce expression of the RstB-His protein, 0.002% (wt/vol) arabinose was added and the culture was reincubated for three additional hours. Parallel batches of pBAD/Myc-HisC-transformed E. coli Top 10 were processed as a negative control. Cells were sedimented by centrifugation for 15 min at 9,000 × g and 4°C and stored at −20°C until processed.The expression of the Orf112 and RstB-His proteins was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cell extracts of VGJφ-infected 569B and E. coli Top 10 transformed with pBAD/Myc-HisC-rstB 9 contained proteins with apparent molecular sizes of 12.7 kDa (Fig. ​(Fig.1)1) and 16 kDa (Fig. ​(Fig.2B),2B), respectively, which are not observed in cells from control cultures. The sizes match those predicted from orf112 (12.72 kDa) (see reference 1) and recombinant rstB-his (16.8 kDa).Open in a separate windowFIG. 1.SDS-PAGE monitoring of the purification process of Orf112 protein. Lane 1, broad-range protein molecular mass markers (Promega); lane 2, cell extract of non-VGJφ-infected V. cholerae 569B; lane 3, cell extract of VGJφ-infected V. cholerae 569B; lane 4, soluble fraction of the sonicate; lane 5, insoluble fraction of the sonicate; lane 6, precipitate at 30% (NH₃)₂SO₄; lane 7, supernatant at 30% (NH₃)₂SO_4; lane 8, precipitate at 50% (NH₃)₂SO₄; lane 9, supernatant at 50% (NH₃)₂SO₄; lane 10, Orf112 protein electro-eluted after preparative SDS-PAGE.Open in a separate windowFIG. 2.Isolation and purification of RstB-His. (A) Chromatogram on a Ni-CAM HC His tag affinity column. (B) SDS-PAGE monitoring of the purification process of RstB-His. Lane 1, broad-range protein molecular mass markers (Promega); lane 2, cell extract of uninduced cultures; lane 3, cell extract of expression-induced cultures; lane 4, soluble fraction of the sonicate from expression-induced cultures; lane 5, insoluble fraction of the sonicate from expression-induced cultures; lane 6, soluble fraction of the 8 M urea extract; lane 7, RstB-His eluted from the column.These proteins are not secreted into the growth medium (not shown); thus, they were released from the cells by ultrasonic disruption as previously described (4). V. cholerae was suspended in 15 ml of 20 mM Tris-HCl buffer, pH 7.5, while E. coli cells were suspended in 15 ml of 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride, pH 8.0. Cell lysates were cleared by centrifugation for 40 min at 9,000 × g and 4°C. SDS-PAGE detected Orf112 protein in the soluble fraction, while RstB-His remained in the insoluble fractions of cell extracts (Fig. ​(Fig.1).1). Subsequently, 569B cell lysate supernatants containing Orf112 protein were fractionated with ammonium sulfate. At 30% ammonium sulfate, several contaminants precipitated but Orf112 protein remained in solution, while at 50% ammonium sulfate, Orf112 precipitated and was recovered by centrifugation. The pellet was washed twice with 50% ammonium sulfate and finally resuspended into 3 ml of 20 mM Tris-HCl buffer, pH 7.5. Removal of excess salt was achieved by gel filtration before the extract was applied to a preparative SDS-PAGE gel. Briefly, a 15% polyacrylamide gel (17 by 19 by 0.5 cm) was run at a constant current intensity of 100 mA and with free voltage at 4°C, until the bromophenol blue dye migrated off the gel. The gels were negatively stained with imidazole-zinc (3), and the Orf112 protein band was identified by comparing bands with the bands of a negative control applied in a neighboring lane of the same gel, where this protein was not visible. The ORF112 protein band was cut from the gel, and the slice was fragmented and introduced into a dialysis bag with a 6- to 8-kDa molecular mass cutoff in 10 ml of 24 mM Tris-HCl-250 mM glycine-0.5% (wt/vol) SDS buffer. The protein was electro-eluted for 5 h at a current intensity of 70 mA and with free voltage at 4°C. Reverse current was applied for 5 min to release membrane-bound proteins, and gel fragments were discarded. The same sample was dialyzed against 1 liter of 0.5 M Tris-HCl, 0.25 M glycine buffer, pH 7.5, for 24 h at 4°C with constant stirring. The dialysis was repeated with 20 mM Tris-HCl, 0.5 M NaCl buffer, pH 7.5, for 24 h. No contaminants were seen when 25 μg of this Orf112 protein-dialyzed extract was checked by SDS-PAGE and Coomassie brilliant blue staining (Fig. ​(Fig.1,1, lane 10).RstB-His protein was recovered from the insoluble fraction of the E. coli lysate by dissolving the lysate in 15 ml of a buffer containing 8 M urea, 20 mM sodium phosphate, 0.5 M NaCl, and 10 mM imidazole, pH 8.0. The mixture was stirred overnight at 4°C and cleared by centrifugation at 9,000 × g for 40 min. The supernatant was applied to a Ni-CAM HC matrix (Sigma), and urea was removed using a linear gradient from 8 to 0 M urea as previously described (5). The presence of 10 mM imidazole in the sample and binding buffer was intended to reduce the level of contaminants bound to the column. Protein was eluted using a gradient of imidazole (10 to 250 mM) in 20 mM sodium phosphate, 0.5 M NaCl buffer, pH 8.0. Fractions were assayed by SDS-PAGE, and those containing the RstB-His protein were pooled according to purity rather than yield. RstB-His protein was obtained with 90% purity (Fig. ​(Fig.2B,2B, lane 7), according to a densitometry scan of Coomassie brilliant blue-stained gels, using a Gene Genius gel documentation system (Syngene Synoptics Ltd., Cambridge, United Kingdom). The gradient-based removal of urea allowed effective solubilization of RstB-His without significant precipitation of protein in the column, as described before (5).Biological activity was assayed by retardation assays of VGJφ genomic ssDNA by 0.5% agarose gel electrophoresis conducted with 20 mM EDTA, 40 mM Tris-acetate buffer. Various amounts of each protein and ssDNA from VGJφ (0.5 μg) mixed in 20% glycerol, 0.25 mM EDTA, 0.3 μM bovine serum albumin, 20 mM Tris-HCl up to a total volume of 40 μl were incubated at room temperature for 30 min and loaded into the gel for analysis. The electrophoresis was run at 100 V and 4°C until colorant exit. Ethidium bromide (1 μg/ml) was used for 30 min to stain DNA bands, which were documented in a Gene Genius gel system (Syngene Synoptics Ltd., Cambridge, United Kingdom).Orf112 protein exhibited DNA retardation activity, showing a high specificity of binding for the circular ssDNA of VGJφ, but was unable to bind double-stranded DNA (dsDNA) (Fig. ​(Fig.3A).3A). However, RstB was able to bind ssDNA as well as dsDNA substrates (Fig. ​(Fig.3B).3B). No retardation was observed with protein preparations from negative controls or when the DNA-protein mixture was inactivated with 1:1 (vol/vol) phenol-chloroform, indicating that the binding activity is intrinsic to the purified proteins.Open in a separate windowFIG. 3.Gel retardation assays of VGJφ-ssDNA by Orf112 protein or RstB-His binding, as measured by 0.5% agarose gel electrophoresis. (A) Binding of Orf112 protein. Lane 1, control of 500 ng of genomic ssDNA of VGJφ; lanes 2 to 6; same as for lane 1 plus 1.25, 2.50, 5.00, 10.0, and 20.0 μg of Orf112 protein, respectively; lane 7, same as for lane 6 but treated with phenol-chloroform; lane 8, linearized replicative-form dsDNA of VGJφ; lane 9, same as for lane 8 plus 20.0 μg of Orf112; lane 10, linearized pUC19; lane 11, same as for lane 10 plus 20.0 μg of Orf112. (B) Binding of RstB-His. Lane 1, control of 500 ng of genomic ssDNA of VGJφ; lane 2, same as for lane 1 plus 20 μg of RstB treated with phenol-chloroform; lanes 3 to 8, same as for lane 1 plus 0.62, 1.25, 2.50, 5.00, 10.0, and 20.0 μg of RstB-His, respectively; lane 9, linearized replicative-form dsDNA of VGJφ; lane 10, same as for lane 9 plus 20.0 μg of RstB-His; lane 11, linearized pUC19; lane 12, same as for lane 11 plus 20.0 μg of RstB-His.In the case of RstB, which binds to ssDNA and dsDNA substrates, we wanted to rule out the possibility that this effect was caused by the His tail fused to the recombinant protein. For this, we recloned RstB in the same vector as RstB-His but without the His tail. We used the same procedure described above for RstB-His but used oligonucleotides CNC06-171 (see above) and CNC06-172 (5′-TACTGCAGTCAAGATTTAATGGGTTG-3′) for RstB amplification. In this case, CNC06-172 introduced a restriction site for PstI, which was used for cloning into pBAD/Myc-HisC.For purification of this RstB variant, E. coli growth and protein expression induction was done as described for RstB-His. Again, RstB was recovered in the insoluble fraction after cell disruption by sonication. Inclusion bodies were resuspended in 15 ml of 50 mM phosphate buffer, pH 7.7, containing 8 M urea, and after overnight stirring at 4°C, the suspension was cleared by centrifugation (9,000 × g, 40 min). The supernatant was applied to an SP Sepharose fast-flow column (Amersham, United Kingdom), and urea was removed as described above for RstB-His. The RstB that bound to the matrix was eluted using a gradient of 0 to 2 M NaCl and was obtained with about 92% purity (data not shown). RstB without the His tail also showed binding activity toward ssDNA and dsDNA substrates (data not shown), ruling out the possibility that the His hexamer is responsible for the nonspecific binding of RstB-His.Since RstB has affinity for both ssDNA and dsDNA, the possibility exists that this protein simply binds any DNA nonspecifically due to an effect of a charge interaction with the phosphate backbone of DNA. In order to study the effect of the charge in the DNA-binding activity of RstB, NaCl was included in the reaction mixture at a concentration from 0 to 500 mM (Fig. ​(Fig.4).4). As can be seen in Fig. ​Fig.4,4, the retardation activity of ssDNA was only partially inhibited from a starting concentration of 400 mM (Fig. ​(Fig.4A),4A), while the retardation of dsDNA started to be partially inhibited at 300 mM NaCl (Fig. ​(Fig.4B).4B). Since RstB continues to bind at high salt concentrations, which should equilibrate the charge effect, these results indicate that the DNA binding activity is not due to the presence of positively charged amino acids in the protein backbone but rather to the presence of domains that specifically recognize the ssDNA or dsDNA.Open in a separate windowFIG. 4.Effect of salt concentration and nonrelated dsDNA competition on the binding of RstB. (A) RstB binding to phage ssDNA in the presence of 0 to 500 mM NaCl as detected by 0.5% agarose gel electrophoresis. Lane 1, 400 ng of genomic ssDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 to 7, same as for lane 2 plus 100, 200, 300, 400, and 500 mM NaCl, respectively. (B) RstB binding to replicative-form phage dsDNA in the presence of 0 to 500 mM NaCl. Lane 1, 400 ng of dsDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 to 7, same as for lane 2 plus 100, 200, 300, 400, and 500 mM NaCl. (C) DNA-binding competition by RstB. Lane 1, 400 ng of genomic ssDNA of VGJφ (control); lane 2, same as for lane 1 plus 15.6 μg of RstB-His; lanes 3 and 4, same as for lane 2 plus 500 and 1,000 ng of sheared calf thymus dsDNA, respectively; lane 5, 500 ng of calf thymus DNA (control).We also investigated whether RstB-His has more affinity for the phage ssDNA than for a nonrelated dsDNA; a DNA-binding competition experiment in which the protein was incubated with a constant amount of ssDNA of VGJφ phage and increasing amounts of calf thymus DNA was performed (Fig. ​(Fig.4C).4C). The ssDNA of VGJφ was retarded by RstB-His even in the presence of 500 and 1,000 ng of calf thymus dsDNA (Fig. ​(Fig.4C,4C, lanes 3 and 4). These results indicate that RstB protein has more affinity for the phage ssDNA than for a non-phage-related dsDNA.Until we know more, RstB is the first protein of a filamentous phage which shows affinity for both ss- and dsDNA, at least in vitro. It is possible that RstB needs another protein from the host or the phage itself to recognize the ssDNA in a specific manner, or perhaps the affinity of RstB for both ss- and dsDNA is an intrinsic property of the protein, which is needed on one hand for binding to genomic ssDNA during the rolling circle replication of the phage and on the other hand for holding the hairpin dsDNA secondary structure formed by the phage genome that functions as the site for integration into the bacterial chromosome (12). This hairpin structure is used by XerCD recombinases as a substrate for recombining the phage genome with the bacterial chromosomal dif site (12), and RstB may act jointly with XerCD to achieve integration. This could explain the requirement of RstB for the integration of CTXφ.It is concluded that Orf112 and RstB proteins purified by the protocols described in this paper were biologically active and obtained at a high degree of purity, which paves the way for further characterization of these proteins. The SSB activities of these two proteins are shown for the first time. Consequently, we propose to rename their respective genes gV^VGJφ and gV^CTXφ and their proteins pV^VGJφ and pV^CTXφ, to follow the denomination of genes of canonical phages of the Inovirus genus. A biochemical and chemical-physical characterization of both proteins is in progress and will be published elsewhere. Should the in vitro role demonstrated for these proteins operate in vivo as well, it might explain their role for rolling circle replication and why rstB is required for CTXφ integration.     

Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae

The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally(inter-strain) and vertically(cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V.cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage's role in the evolutionary and epidemiological mechanisms of V. cholerae.     

Survival of Vibrio cholerae in Nutrient-Poor Environments Is Associated with a Novel “Persister” Phenotype

In response to antibiotic and/or environmental stress, some species of bacteria shift to a “persister” phenotype. Although toxigenic Vibrio cholerae, responsible for the disease cholera, can be found in nutrient-poor aquatic environments in endemic areas, the underlying mechanism(s) by which culturable cells persist in these environmental reservoirs is largely unknown. Here we report that introduction of V. cholerae into a nutrient-poor filter sterilized lake water (FSLW) microcosm promoted a shift to what we have defined as a “persister” phenotype (PP) which was culturable for >700 days. Direct transfer of PP of V. cholerae from original microcosms to freshly prepared FSLW resulted in the same pattern of persistence seen in the original microcosms. Scanning electron microscopy of cells persisting for over 700 days demonstrated cell morphologies that were very small in size, with a high degree of aggregation associated with flagella emanating from all aspects of the cell. V. cholerae PP cells reverted to a typical V. cholerae morphology when transferred to nutrient-rich L- broth. Cell-free supernatants obtained from microcosms at 24 hours, 180 days, and 700 days all showed >2-fold increase in CAI-1 signaling molecules, consistent with quorum sensing activity, as has been described for Pseudomonas aeruginosa persister cells. Chitin and phosphate promoted cell growth. Our data suggest that nutrient stress can select a V. cholerae persister phenotype in environmental reservoirs, with these strains then seeding subsequent cholera epidemics in response to chitin and phosphate availability.     

Crystal Structures of β-Primeverosidase in Complex with Disaccharide Amidine Inhibitors

β-Primeverosidase (PD) is a disaccharide-specific β-glycosidase in tea leaves. This enzyme is involved in aroma formation during the manufacturing process of oolong tea and black tea. PD hydrolyzes β-primeveroside (6-O-β-d-xylopyranosyl-β-d-glucopyranoside) at the β-glycosidic bond of primeverose to aglycone, and releases aromatic alcoholic volatiles of aglycones. PD only accepts primeverose as the glycone substrate, but broadly accepts various aglycones, including 2-phenylethanol, benzyl alcohol, linalool, and geraniol. We determined the crystal structure of PD complexes using highly specific disaccharide amidine inhibitors, N-β-primeverosylamidines, and revealed the architecture of the active site responsible for substrate specificity. We identified three subsites in the active site: subsite −2 specific for 6-O-β-d-xylopyranosyl, subsite −1 well conserved among β-glucosidases and specific for β-d-glucopyranosyl, and wide subsite +1 for hydrophobic aglycone. Glu-470, Ser-473, and Gln-477 act as the specific hydrogen bond donors for 6-O-β-d-xylopyranosyl in subsite −2. On the other hand, subsite +1 was a large hydrophobic cavity that accommodates various aromatic aglycones. Compared with aglycone-specific β-glucosidases of the glycoside hydrolase family 1, PD lacks the Trp crucial for aglycone recognition, and the resultant large cavity accepts aglycone and 6-O-β-d-xylopyranosyl together. PD recognizes the β-primeverosides in subsites −1 and −2 by hydrogen bonds, whereas the large subsite +1 loosely accommodates various aglycones. The glycone-specific activity of PD for broad aglycone substrates results in selective and multiple release of temporally stored alcoholic volatile aglycones of β-primeveroside.     

Structures of REV1 UBM2 Domain Complex with Ubiquitin and with a Small-Molecule that Inhibits the REV1 UBM2–Ubiquitin Interaction

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2–ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.     

Crystal Structures of the Free and Ligand-Bound FK1–FK2 Domain Segment of FKBP52 Reveal a Flexible Inter-Domain Hinge

The human Hsp90 co-chaperone FKBP52 belongs to the family of FK506-binding proteins, which act as peptidyl-prolyl isomerases. FKBP52 specifically enhances the signaling of steroid hormone receptors, modulates ion channels and regulates neuronal outgrowth dynamics. In turn, small-molecule ligands of FKBP52 have been suggested as potential neurotrophic or anti-prostate cancer agents. The usefulness of available ligands is however limited by a lack of selectivity. The immunophilin FKBP52 is composed of three domains, an FK506-binding domain with peptidyl-prolyl isomerase activity, an FKBP-like domain of unknown function and a TPR-clamp domain, which recognizes the C-terminal peptide of Hsp90 with high affinity. The herein reported crystal structures of FKBP52 reveal that the short linker connecting the FK506-binding domain and the FKBP-like domain acts as a flexible hinge. This enhanced flexibility and its modulation by phosphorylation might explain some of the functional antagonism between the closely related homologs FKBP51 and FKBP52. We further present two co-crystal structures of FKBP52 in complex with the prototypic ligand FK506 and a synthetic analog thereof. These structures revealed the molecular interactions in great detail, which enabled in-depth comparison with the corresponding complexes of the other cytosolic FKBPs, FKBP51 and FKBP12. The observed subtle differences provide crucial insights for the rational design of ligands with improved selectivity for FKBP52.     

Crystal Structure of an EAL Domain in Complex with Reaction Product 5′-pGpG

FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438–686): one of the apo form and the other of a complex with 5′-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5′-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.     

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+?Calmodulin Displays a Unique Binding Mode

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca²⁺-depletion sensor STIM1. This is followed by a fast Ca²⁺·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp⁷⁶ of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.     

The Structure of Integrin α1I Domain in Complex with a Collagen-mimetic Peptide

We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.     

Neutron and X-ray Crystal Structures of a Perdeuterated Enzyme Inhibitor Complex Reveal the Catalytic Proton Network of the Toho-1 β-Lactamase for the Acylation Reaction

The mechanism by which class A β-lactamases hydrolyze β-lactam antibiotics has been the subject of intensive investigation using many different experimental techniques. Here, we report on the novel use of both neutron and high resolution x-ray diffraction to help elucidate the identity of the catalytic base in the acylation part of the catalytic cycle, wherein the β-lactam ring is opened and an acyl-enzyme intermediate forms. To generate protein crystals optimized for neutron diffraction, we produced a perdeuterated form of the Toho-1 β-lactamase R274N/R276N mutant. Protein perdeuteration, which involves replacing all of the hydrogen atoms in a protein with deuterium, gives a much stronger signal in neutron diffraction and enables the positions of individual deuterium atoms to be located. We also synthesized a perdeuterated acylation transition state analog, benzothiophene-2-boronic acid, which was also isotopically enriched with ¹¹B, as ¹⁰B is a known neutron absorber. Using the neutron diffraction data from the perdeuterated enzyme-inhibitor complex, we were able to determine the positions of deuterium atoms in the active site directly rather than by inference. The neutron diffraction results, along with supporting bond-length analysis from high resolution x-ray diffraction, strongly suggest that Glu-166 acts as the general base during the acylation reaction.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

Prediction of Protein-Protein Interfaces on G-Protein β Subunits Reveals a Novel Phospholipase C β2 Binding Domain

Gβ subunits from heterotrimeric G-proteins (guanine nucleotide-binding proteins) directly bind diverse proteins, including effectors and regulators, to modulate a wide array of signaling cascades. These numerous interactions constrained the evolution of the molecular surface of Gβ. Although mammals contain five Gβ genes comprising two classes (Gβ1-like and Gβ5-like), plants and fungi have a single ortholog, and organisms such as Caenorhabditis elegans and Drosophila melanogaster contain one copy from each class. A limited number of crystal structures of complexes containing Gβ subunits and complementary biochemical data highlight specific sites within Gβs needed for protein interactions. It is difficult to determine from these interaction sites what, if any, additional regions of the Gβ molecular surface comprise interaction interfaces essential to Gβ's role as a nexus in numerous signaling cascades. We used a comparative evolutionary approach to identify five known and eight previously unknown putative interfaces on the surface of Gβ. We show that one such novel interface occurs between Gβ and phospholipase C β2 (PLC-β2), a mammalian Gβ interacting protein. Substitutions of residues within this Gβ-PLC-β2 interface reduce the activation of PLC-β2 by Gβ1, confirming that our de novo comparative evolutionary approach predicts previously unknown Gβ-protein interfaces. Similarly, we hypothesize that the seven remaining untested novel regions contribute to putative interfaces for other Gβ interacting proteins. Finally, this comparative evolutionary approach is suitable for application to any protein involved in a significant number of protein-protein interactions.     

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

Background

Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL).

Methodology/Principal Findings

We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure.

Conclusion

Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.     

Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties

We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.