DNase Domain Research Articles

Application of a Novel CL7/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins.

We have developed the CL7/Im7 protein purification system to achievehigh-yield, high-purity and high-activity (HHH) products in one step. The system is based on the natural ultrahigh-affinity complex between the two small proteins encoded by colicinogenic plasmids carried by certain E. coli strains, the DNAse domain of colicin E7 (CE7; MW~15kDa) and its natural endogenous inhibitor, the immunity protein 7 (Im7; MW~10kDa). CL7 is an engineered variant of CE7, in which the toxic DNA-binding and catalytic activities have been eliminated while retaining the high affinity to Im7. CL7 is used as a protein tag, while Im7 is covalently attached to agarose beads. To make the CL7/Im7 technique easy to use, we have designed a set of the E. coli expression vectors for fusion of a target protein to the protease-cleavableCL7-tag either at the N- or the C-terminus, and also have the options of the dual (CL7/His8) tag. A subset of vectors is dedicated for cloning membrane and multisubunit proteins. The CL7/Im7 system has severalnotable advatantages over other available affinity purification techniques. First, high concentrations of the small Im7 protein are coupled to the beads resulting in the high column capacities (up to 60mg/mL). Second, an exceptional stability of Im7 allows for multiple (100+) regeneration cycles with no loss of binding capacities. Third, the CL7-tag improves protein expression levels, solubility and, in some cases, assists folding of the target proteins. Fourth, the on-column proteolytic elution produces purified proteins with few or no extra amino acid residues. Finally, the CL7/Im7 affinity is largely insensitive to high salt concentrations. For many target proteins, loading the bacterial lysates on the Im7 column in high salt is a key to high purity. Altogether, these properties of the CL7/Im7 system allow for a one-step HHH purification of most challenging, biologically and clinically significant proteins.

Overexpression of lncRNA TATDN1 Promotes Cancer Cell Proliferation in Triple Negative Breast Cancer by Regulating miR-26b Methylation.

BackgroundLong non-coding RNA (lncRNA) TatD DNase Domain Containing 1 (TATDN1) is a recently characterized oncogenic lncRNA in several types of cancer including breast cancer. Our preliminary microarray analysis revealed its upregulation in triple negative breast cancer (TNBC) and its inverse correlation with microRNA-26b (miR-26b), which is a tumor suppressive miRNA in breast cancer. This study was therefore carried out to investigate the interaction between TATDN1 and miR-26b in TNBC.MethodsA total of 66 pairs of TNBC and non-tumor tissues were collected from 66 patients (45.8 ± 10.5 years old) with TNBC through biopsy under the guidance of MRI before initiation of any therapies. Quantitative reverse transcription PCR (RT-qPCR), transient cell transfection, methylation specific PCR (MSP) and cell proliferation assay were carried out in this study.ResultsWe found that TATDN1 was upregulated and miR-26b was downregulated in TNBC. Correlation analysis showed that the expression of TATDN1 and miR-26b was inversely correlated. In TNBC cells, overexpression of TATDN1 mediated the downregulation of miR-26b. Knockdown of TATDN1 led to the upregulation of miR-26b. Methylation-specific PCR showed that TATDN1 positively regulated the methylation of miR-26b gene. Cell proliferation analysis showed that TATDN1 positively regulated the proliferation of TNBC cells. Overexpression of miR-26b attenuated the effects of TATDN1 overexpression on cell proliferation.ConclusionTherefore, overexpression of TATDN1 promotes cancer cell proliferation in TNBC by regulating the methylation of miR-26b gene.

A modular effector with a DNase domain and a marker for T6SS substrates

Bacteria deliver toxic effectors via type VI secretion systems (T6SSs) to dominate competitors, but the identity and function of many effectors remain unknown. Here we identify a Vibrio antibacterial T6SS effector that contains a previously undescribed, widespread DNase toxin domain that we call PoNe (Polymorphic Nuclease effector). PoNe belongs to a diverse superfamily of PD-(D/E)xK phosphodiesterases, and is associated with several toxin delivery systems including type V, type VI, and type VII. PoNe toxicity is antagonized by cognate immunity proteins (PoNi) containing DUF1911 and DUF1910 domains. In addition to PoNe, the effector contains a domain of unknown function (FIX domain) that is also found N-terminal to known toxin domains and is genetically and functionally linked to T6SS. FIX sequences can be used to identify T6SS effector candidates with potentially novel toxin domains. Our findings underline the modular nature of bacterial effectors harboring delivery or marker domains, specific to a secretion system, fused to interchangeable toxins.

"RETRACTED ARTICLE: Vibrio parahaemolyticus RhsP represents a widespread group of pro-effectors for type VI secretion systems

Type VI secretion systems (T6SSs) translocate effector proteins, such as Rhs toxins, to eukaryotic cells or prokaryotic competitors. All T6SS Rhs-type effectors characterized thus far contain a PAAR motif or a similar structure. Here, we describe a T6SS-dependent delivery mechanism for a subset of Rhs proteins that lack a PAAR motif. We show that the N-terminal Rhs domain of protein RhsP (or VP1517) from Vibrio parahaemolyticus inhibits the activity of the C-terminal DNase domain. Upon auto-proteolysis, the Rhs fragment remains inside the cells, and the C-terminal region interacts with PAAR2 and is secreted by T6SS2; therefore, RhsP acts as a pro-effector. Furthermore, we show that RhsP contributes to the control of certain “social cheaters” (opaR mutants). Genes encoding proteins with similar Rhs and PAAR-interacting domains, but diverse C-terminal regions, are widely distributed among Vibrio species.

Mitotic Phosphorylation of TREX1 C Terminus Disrupts TREX1 Regulation of the Oligosaccharyltransferase Complex

SUMMARYTREX1 mutations are associated with several auto-immune and inflammatory diseases. The N-terminal DNase domain of TREX1 is important for preventing self-DNA from activating the interferon response. The C terminus of TREX1 is required for ER localization and regulation of oligosacchariyltransferase (OST) activity. Here, we show that during mitosis TREX1 is predominately phosphorylated at the C-terminal Serine-261 by Cyclin B/CDK1. TREX1 is dephosphorylated quickly at mitotic exit, likely by PP1/PP2-type serine/threonine phosphatase. Mitotic phosphorylation does not affect TREX1 DNase activity. Phosphomimetic mutations of mitotic phosphorylation sites in TREX1 disrupted the interaction with the OST subunit RPN1. RNA-seq analysis of Trex1−/− mouse embryonic fibroblasts expressing TREX1 wild-type or phosphor-mutants revealed a glycol-gene signature that is elevated when TREX1 mitotic phosphorylation sites are disrupted. Thus, the cell-cycle-dependent post-translation modification of TREX1 regulates its interaction with OST, which may have important implications for immune disease associated with the DNase-independent function of TREX1.

On the ability of molecular dynamics simulation and continuum electrostatics to treat interfacial water molecules in protein-protein complexes

Interfacial waters are increasingly appreciated as playing a key role in protein-protein interactions. We report on a study of the prediction of interfacial water positions by both Molecular Dynamics and explicit solvent-continuum electrostatics based on the Dipolar Poisson-Boltzmann Langevin (DPBL) model, for three test cases: (i) the barnase/barstar complex (ii) the complex between the DNase domain of colicin E2 and its cognate Im2 immunity protein and (iii) the highly unusual anti-freeze protein Maxi which contains a large number of waters in its interior. We characterize the waters at the interface and in the core of the Maxi protein by the statistics of correctly predicted positions with respect to crystallographic water positions in the PDB files as well as the dynamic measures of diffusion constants and position lifetimes. Our approach provides a methodology for the evaluation of predicted interfacial water positions through an investigation of water-mediated inter-chain contacts. While our results show satisfactory behaviour for molecular dynamics simulation, they also highlight the need for improvement of continuum methods.

Characterization of a DUF820 family protein Alr3200 of the cyanobacterium Anabaena sp. strain PCC7120

The hypothetical protein 'Alr3200' of Anabaena sp. strain PCC7120 is highly conserved among cyanobacterial species. It is a member of the DUF820 (Domain of Unknown Function) protein family, and is predicted to have a DNase domain. Biochemical analysis revealed a Mg(II)-dependent DNase activity for Alr3200 with a specific activity of 8.62x104 Kunitz Units (KU) mg -1 protein. Circular dichroism analysis predicted Alr3200 to have approximately 40 percent beta-strands and approximately 9 percent alpha-helical structures. Anabaena PCC7120 inherently expressed Alr3200 at very low levels, and its overexpression had no significant effect on growth of Anabaena under control conditions. However, Analr3200+, the recombinant Anabaena strain overexpressing Alr3200, exhibited zero survival upon exposure to 6 kGy of gamma-radiation, which is the LD50 for wild type Anabaena PCC7120 as well as the vector control recombinant strain, AnpAM. Comparative analysis of the two recombinant Anabaena strains suggested that it is not the accumulated Alr3200 per se, but its possible interactions with the radiation-induced unidentified DNA repair proteins of Anabaena, which hampers DNA repair resulting in radiosensitivity.

The role and potential mechanisms of LncRNA-TATDN1 on metastasis and invasion of non-small cell lung cancer.

The invasion and metastasis of malignant tumor cells lead to normal tissue destruction and are major prognostic factors for many malignant cancers. Long non-coding RNA (LncRNA) is associated with occurrence, development and prognoses of non-small cell lung cancer (NSCLC), but its mechanisms of action involved in tumor invasion and metastasis are not clear. In this study, we screened and detected the expression of LncRNA in two NSCLC lines 95D and 95C by using high throughput LncRNA chip. We found that TATDN1 (Homo sapiens TatD DNase domain containing 1, TATDN1), one of LncRNAs, was highly expressed in 95D cells and NSCLC tumor tissues compared to 95C cells. Knockdown of TATDN1–1 by shRNA significantly inhibited cell proliferation, adhesion, migration and invasion in 95D cells. Further mechanism study showed that TATDN1 knockdown suppressed the expression of E-cadherin, HER2, β-catenin and Ezrin. Moreover, knockdown TATDN1 also inhibited tumor growth and metastasis in a 95D mouse model in vivo by inhibiting β-catenin and Ezrin. These data indicate that TATDN1 expression is associated with 95D cells' higher potential of invasion and metastasis, and suggest that TATDN1 may be a potential prognostic factor and therapeutic target for NSCLCs.

Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups.

In vivo processing of DNase colicins E2 and E7 is required for their import into the cytoplasm of target cells.

DNase colicins E2 and E7, both of which appropriate the BtuB/Tol translocation machinery to cross the outer membrane, undergo a processing step as they enter the cytoplasm. This endoproteolytic cleavage is essential for their killing action. A processed form of the same size, 18.5 kDa, which corresponds to the C-terminal catalytic domain, was detected in the cytoplasm of bacteria treated with either of the two DNase colicins. The inner-membrane protease FtsH is necessary for the processing that allows the translocation of the colicin DNase domain into the cytoplasm. The processing occurs near residue D420, at the same position as the FtsH-dependent cleavage in RNase colicins E3 and D. The cleavage site is located 30 amino acids upstream of the DNase domain. In contrast, the previously reported periplasm-dependent colicin cleavage, located at R452 in colicin E2, was shown to be generated by the outer-membrane protease OmpT and we show that this cleavage is not physiologically relevant for colicin import. Residue R452, whose mutated derivatives led to toxicity defect, was shown to have no role in colicin processing and translocation, but it plays a key role in the catalytic activity, as previously reported for other DNase colicins. Membrane associated forms of colicins E2 and E7 were detected on target cells as proteinase K resistant peptides, which include both the receptor-binding and DNase domains. A similar, but much less proteinase K-resistant form was also detected with RNase colicin E3. These colicin forms are not relevant for colicin import, but their detection on the cell surface indicates that whole nuclease-colicin molecules are found in a stable association with the outer-membrane receptor BtuB of the target cells.

Blind prediction of interfacial water positions in CAPRI

We report the first assessment of blind predictions of water positions at protein-protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions-20 groups submitted a total of 195 models-were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models-a very good docking performance per se-only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein-water interactions and their role in stabilizing protein complexes.

Extending RosettaDock with water, sugar, and pH for prediction of complex structures and affinities for CAPRI rounds 20-27.

Rounds 20-27 of the Critical Assessment of PRotein Interactions (CAPRI) provided a testing platform for computational methods designed to address a wide range of challenges. The diverse targets drove the creation of and new combinations of computational tools. In this study, RosettaDock and other novel Rosetta protocols were used to successfully predict four of the 10 blind targets. For example, for DNase domain of Colicin E2-Im2 immunity protein, RosettaDock and RosettaLigand were used to predict the positions of water molecules at the interface, recovering 46% of the native water-mediated contacts. For α-repeat Rep4-Rep2 and g-type lysozyme-PliG inhibitor complexes, homology models were built and standard and pH-sensitive docking algorithms were used to generate structures with interface RMSD values of 3.3 Å and 2.0 Å, respectively. A novel flexible sugar-protein docking protocol was also developed and used for structure prediction of the BT4661-heparin-like saccharide complex, recovering 71% of the native contacts. Challenges remain in the generation of accurate homology models for protein mutants and sampling during global docking. On proteins designed to bind influenza hemagglutinin, only about half of the mutations were identified that affect binding (T55: 54%; T56: 48%). The prediction of the structure of the xylanase complex involving homology modeling and multidomain docking pushed the limits of global conformational sampling and did not result in any successful prediction. The diversity of problems at hand requires computational algorithms to be versatile; the recent additions to the Rosetta suite expand the capabilities to encompass more biologically realistic docking problems.

Immunity protein release from a cell‐bound nuclease colicin complex requires global conformational rearrangement

Nuclease colicins bind their target receptor BtuB in the outer membrane of sensitive Escherichia coli cells in the form of a high-affinity complex with their cognate immunity proteins. The release of the immunity protein from the colicin complex is a prerequisite for cell entry of the colicin and occurs via a process that is still relatively poorly understood. We have previously shown that an energy input in the form of the cytoplasmic membrane proton motive force is required to promote immunity protein (Im9) release from the colicin E9/Im9 complex and colicin cell entry. We report here that engineering rigidity in the structured part of the colicin translocation domain via the introduction of disulfide bonds prevents immunity protein release from the colicin complex. Reduction of the disulfide bond by the addition of DTT leads to immunity protein release and resumption of activity. Similarly, the introduction of a disulfide bond in the DNase domain previously shown to abolish channel formation in planar bilayers also prevented immunity protein release. Importantly, all disulfide bonds, in the translocation as well as the DNase domain, also abolished the biological activity of the Im9-free colicin E9, the reduction of which led to a resumption of activity. Our results show, for the first time, that conformational flexibility in the structured translocation and DNase domains of a nuclease colicin is essential for immunity protein release, providing further evidence for the hypothesis that global structural rearrangement of the colicin molecule is required for disassembly of this high-affinity toxin-immunity protein complex prior to outer membrane translocation.

A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

Author SummaryMany proteins interact with other proteins as part of their function. One method of modulating the activity of protein complexes is to break them apart. Some complexes, however, are extremely kinetically stable and it is unclear how these can dissociate on a biologically relevant timescale. In this study we address this question using protein complexes between colicin E9 (a bacterial toxin) and its immunity protein Im9. These highly avid complexes (with a lifetime of days) must be broken apart for colicin to be activated. By using single-molecule force methods we show that pulling on one end of colicin E9 drastically destabilises the complex so that it dissociates a million-fold faster than its intrinsic rate. We then show that preventing this destabilisation (by the insertion of cross-links that pin the N-terminus of E9 in place) yields a kinetically stable complex. It has previously been postulated that force can destabilise a protein complex by partially unfolding one or more binding partners. Our work provides new experimental evidence that shows this is the case and provides a mechanism for this phenomenon, which we term a trip bond. For the E9:Im9 complex, trip bond behaviour allows a stable complex to be rapidly dissociated by application of a surprisingly small force.

Interaction of Nuclease Colicins with Membranes: Insertion Depth Correlates with Bilayer Perturbation

BackgroundProtein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases) or RNA (RNases) hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7) and one ribosomal RNase domain (E3) using a biomembrane model system.Principal ResultsWe demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed.Conclusions/SignificanceOur results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.

Association of Gene-Gene Interactions with Venous Thromboembolism (VTE): A Merged/Imputed Genome-Wide Scan/Candidate-Gene Case-Control Study

Abstract 1242 Background:While bivariate interactions between Factor V Leiden, prothrombin G20201A and hereditary deficiency of antithrombin, protein C and protein S compound VTE risk, whether other gene-gene interactions are associated with VTE is largely unknown. Objective:To test novel gene-gene interactions for an association with VTE. Methods:Genome-wide scan (Illumina 660Q [557,112 SNPs]) and candidate gene (12,551 SNPs in 764 genes within the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways) genotypes from 1270 non-Hispanic adults of European ancestry with objectively-diagnosed VTE (cases; no cancer, venous catheter or antiphospholipid antibodies) and 1302 controls (frequency-matched on case age, gender, race, MI/stroke status) were merged and imputed to about 2.5 million SNPs with MACH using HapMap Phase 2 (60 CEU). We tested all pairwise SNP-SNP interactions using a 2-stage procedure (fast epistasis in the first stage and age, gender, MI/stroke status and state of residence adjusted analysis using logistic regression in the second stage). Results:The mean ± standard deviation case and control ages were 54.4 ± 16.2 and 55.6 ± 15.8 years, respectively and 51% were female. Among 2.299×10^12 pairwise interactions tested, 900 million reached a p-value<E-06 and were be tested in the second stage. Of these, 2417, 278 and 22 SNP-SNP interactions reached p-values ≤1E-9, 1E–10, and1E-11 respectively; 121 SNP-SNP interactions reached the Bonferroni statistical significance threshold (≤5.555659E-11). The gene-gene pairwise interactions with p-values≤1E-11 were: (1) chromosome 3 open reading frame 70 (C3orf70) – short-chain dehydrogenase/reductase (SDR family) member 4 (DHRS4), (2) 1-acylglycerol-3-phosphate O-acyltransferase 4 (AGPAT4) - NIMA (never in mitosis gene a)-related kinase 6 (NEK6), and (3) TatD DNase domain containing 2 (TATDN2) – WAS protein family, member 3 (WASF3). No gene function data are available for C3orf70. DHRS4 encodes for a 3-ketosteroid reductase important for active steroid hormone regulation, AGPAT4 is important for signal transduction and lipid biosynthesis, NEK6 encodes for Nek6 which downregulates p53-induced cancer cell senescence, TATDN2 encodes a deoxyribonuclease, WASF3 encodes for a member of the Wiskott-Aldrich syndrome protein family which are key regulators of signal transduction and cytoskeletal reorganization in hematopoietic cells. Conclusion:Pairwise gene-gene interaction analyses suggest potential associations between VTE and novel genes for future testing in replication studies. Disclosures:Heit:Daiichi Sankyo: Consultancy, Honoraria.

NMR Characterisation of the Relationship between Frustration and the Excited State of Im7

Previous work shows that Im9 folds in a two-state transition while its homologue Im7 folds in a three-state transition via an on-pathway kinetic intermediate state (KIS), with this difference being related to frustration in the structure of Im7. We have used NMR spectroscopy to study conformational dynamics connected to the frustration. A combination of equilibrium peptide N 1H/N 2H exchange, model-free analyses of backbone NH relaxation data and relaxation dispersion (RD)-NMR shows that the native state of Im7 is in equilibrium with an intermediate state that is lowly populated [equilibrium intermediate state (EIS)]. Comparison of kinetic and thermodynamic parameters describing the EIS native-state equilibrium obtained by RD-NMR with previously reported parameters describing the KIS native-state equilibrium obtained from stopped-flow fluorescence studies of refolding His-tagged Im7 shows that the KIS and the EIS are the same species. 15N chemical shifts of the EIS obtained from the RD-NMR analysis show that residues forming helix III in the native state are unstructured in the EIS while other residues experiencing frustration in the native state are in structured regions of the EIS. We show that binding of Im7 and its L53A/I54A variant (which resembles the EIS as shown in previous work) to the cognate partner for Im7, the DNase domain of colicin E7, causes the dynamic processes associated with the frustration to be dampened.

Characterization of a New Bacteriocin, Carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21

Two different bacteriocins, carotovoricin and carocin S1, had been found in Pectobacterium carotovorum subsp. carotovorum, which causes soft-rot disease in diverse plants. Previously, we reported that the particular strain Pcc21, producing only one high-molecular-weight bacteriocin, carried a new antibacterial activity against the indicator strain Pcc3. Here, we report that this new antibacterial activity is due to a new bacteriocin produced by strain Pcc21 and named carocin D. Carocin D is encoded by the caroDK gene located in the genomic DNA together with the caroDI gene, which seems to encode an immunity protein. N-terminal amino acid sequences of purified carocin D were determined by Edman degradation. In comparison with the primary translation product of caroDK, it was found that 8 amino acids are missing at the N terminus. This finding proved that carocin D is synthesized as a precursor peptide and that 8 amino acids are removed from its N terminus during maturation. Carocin D has two putative translocation domains; the N-terminal and C-terminal domains are homologous to those of Escherichia coli colicin E3 and Pseudomonas aeruginosa S-type pyocin, respectively. When caroDK and caroDI genes were transformed into carocin D-sensitive bacteria such as Pcc3, the bacteria became resistant to this bacteriocin. Carocin D has one putative DNase domain at the extreme C terminus and showed DNase activity in vitro. This bacteriocin had slight tolerance to heat but not to proteases. The caroDK gene was present in only 5 of 54 strains of P. carotovorum subsp. carotovorum. These results indicate that carocin D is a third bacteriocin found in P. carotovorum subsp. carotovorum, and this bacteriocin can be readily expressed in carocin D-sensitive nonpathogenic bacteria, which may have high potential as a biological control agent in the field.

Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin

We have developed a polypeptide lysostaphin FRET (fluorescence resonance energy transfer) substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at positions 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue at each of four positions of the pentaglycine target site and it was found that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP (green fluorescent protein) domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay, the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.

Kinetic screening of antibody–Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V NAR single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors.