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The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.

Colicin E3 is a 60 kDa, multidomain protein antibiotic that targets its ribonuclease activity to an essential region of the 16S ribosomal RNA of Escherichia coli. To prevent suicide of the producing cell, synthesis of the toxin is accompanied by the production of a 10 kDa immunity protein (Im3) that binds strongly to the toxin and abolishes its enzymatic activity. In the present work, we study the interaction of Im3 with the isolated cytotoxic domain (E3 rRNase) and intact colicin E3 through presteady-state kinetics and thermodynamic measurements. The isolated E3 rRNase domain forms a high affinity complex with Im3 (K(d) = 10(-12) M, in 200 mM NaCl at pH 7.0 and 25 degrees C). The interaction of Im3 with full-length colicin E3 under the same conditions is however significantly stronger (K(d) = 10(-14) M). The difference in affinity arises almost wholly from a marked decrease in the dissociation rate constant for the full-length complex (8 x 10(-7) s(-1)) relative to the E3 rRNase-Im3 complex (1 x 10(-4) s(-1)), with their association rates comparable ( approximately 10(8) M(-1) s(-1)). Thermodynamic measurements show that complex formation is largely enthalpy driven. In light of the recently published crystal structure of the colicin E3-Im3 complex, the additional stabilization of the wild-type complex can be ascribed to the interaction of Im3 with the N-terminal translocation domain of the toxin. These observations suggest a mechanism whereby dissociation of the immunity protein prior to translocation into the target cell is facilitated by the loss of the Im3-translocation domain interaction.

Colicin E9 is a 61 kDa antibacterial protein secreted by E. coli. In order for it to enter the cytoplasm of susceptible bacteria and kill them by hydrolysing their DNA, the colicin must first interact with an outer membrane receptor on the target cell, BtuB, and a translocation pathway involving Tol proteins. The receptor binding, translocation and DNase functions of colicin E9 are housed in discrete structural domains, which have been independently expressed and characterized. The minimal receptor-binding domain is a 76 amino acid protein (min-R). X-ray structure determination of a related colicin shows its receptor-binding-domain to have a helical hairpin structure (S. Soelaiman, K. Jakes, N. Wu, C. Li and M. Shoham, Molecular Cell. 2001, 8, 1053). Our solution NMR studies of min-R have confirmed it has a helical hairpin structure, and shown it has multiple slowly interchanging conformers and a flexible inter-helix loop. A plausible interpretation of these data is that in solution the helical hairpin can adopt a variety of structures differing in the spatial relationship of the two helices. A possible biological role for this involves the hairpin opening during translocation into bacteria.

The extracytoplasmic function (ECF) sigma factor sigma(R) is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sigma(R) dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sigma(R) or how it suppresses sigma(R)-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, approximately 10kDa domain (sigma(RN)) that encompasses region 2 of sigma(R) that represents the major RsrA binding site. We show that sigma(RN) inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits sigma(R) activity is by blocking this association. We also report the 2.4A resolution crystal structure of sigma(RN) that reveals extensive structural conservation with the equivalent region of sigma(70) from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. sigma(RN) has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.

We report the first stopped-flow fluorescence analysis of transition metal binding (Co(2+), Ni(2+), Cu(2+), and Zn(2+)) to the H-N-H endonuclease motif within colicin E9 (the E9 DNase). The H-N-H consensus forms the active site core of a number of endonuclease groups but is also structurally homologous to the so-called treble-clef motif, a ubiquitous zinc-binding motif found in a wide variety of metalloproteins. We find that all the transition metal ions tested bind via multistep mechanisms. Binding was further dissected for Ni(2+) and Zn(2+) ions through the use of E9 DNase single tryptophan mutants, which demonstrated that most steps reflect conformational rearrangements that occur after the bimolecular collision, many common to the two metals, while one appears specific to zinc. The kinetically derived equilibrium dissociation constants (K(d)) for transition metal binding to the E9 DNase agree with previously determined equilibrium measurements and so confirm the validity of the derived kinetic mechanisms. Zn(2+) binds tightest to the enzyme (K(d) approximately 10(-)(9) M) but does not support endonuclease activity, whereas the other metals (K(d) approximately 10(-)(6) M) are active in endonuclease assays implying that the additional step seen for Zn(2+) traps the enzyme in an inactive but high affinity state. Metal-induced conformational changes are likely to be a conserved feature of H-N-H/treble clef motif proteins since similar Zn(2+)-induced, multistep binding was observed for other colicin DNases. Moreover, they appear to be independent both of the conformational heterogeneity that is naturally present within the E9 DNase at equilibrium, as well as the conformational changes that accompany the binding of its cognate inhibitor protein Im9.

Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H-N-H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H-N-H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H-N-H motif is the site of DNA binding and that Mg2+-dependent cleavage of double-stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation-dependent catalytic mechanisms. The roles of individual amino acids within the H-N-H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase-activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H-N-H DNases.

Nano-electrospray ionization time-of-flight mass spectrometry (ESI-MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI-MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far- and near-UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI-MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid-induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI-MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn(2+). Gas-phase dissociation experiments of the deuterium-labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas-phase dissociation experiments of the ternary E9 DNase-Zn(2+)-Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself., (c) 2002 Elsevier Science Ltd.)

The process by which the endonuclease domain of colicin E9 is translocated across the outer membrane, the periplasmic space and the cytoplasmic membrane to reach the cytoplasm of E. coli cells, resulting in DNA degradation and cell death, is a unique event in prokaryotic biology. Although considerable information is known about the role of the BtuB outer membrane receptor, as well as the mostly periplasmic Tol proteins that are essential for the translocation process, the precise nature of the interactions between colicin E9 and these proteins remains to be elucidated. In this review, we consider our current understanding of the key events in this process, concentrating on recent findings concerning receptor-binding, translocation and the mechanism of cytotoxicity.

Nuclease E colicins that exert their cytotoxic activity through either non-specific DNase or specific rRNase action are inhibited by immunity proteins in a high affinity interaction that gives complete protection to the producing host cell from the deleterious effects of the toxin. Previous X-ray crystallographic analysis of these systems has revealed that in both cases, the immunity protein inhibitor forms its highly stable complex with the enzyme by binding as an exosite inhibitor-adjacent to, but not obscuring, the enzyme active site. The structures of the free E9 DNase domain and its complex with an ssDNA substrate now show that inhibition is achieved without deformation of the enzyme and by occlusion of a limited number of residues of the enzyme critical in recognition and binding of the substrate that are 3' to the cleaved scissile phosphodiester. No sequence or structural similarity is evident between the two classes of cytotoxic domain, and the heterodimer interfaces are also dissimilar. Thus, whilst these structures suggest the basis for specificity in each case, they give few indications as to the basis for the remarkably strong binding that is observed. Structural analyses of complexes bearing single site mutations in the immunity protein at the heterodimer interface reveal further differences. For the DNases, a largely plastic interface is suggested, where optimal binding may be achieved in part by rigid body adjustment in the relative positions of inhibitor and enzyme. For the rRNases, a large solvent-filled cavity is found at the immunity-enzyme interface, suggesting that other considerations, such as that arising from the entropy contribution from bound water molecules, may have greater significance in the determination of rRNase complex affinity than for the DNases.

Many proteins populate collapsed intermediate states during folding. In order to elucidate the nature and importance of these species, we have mapped the structure of the on-pathway intermediate of the four-helix protein, Im7, together with the conformational changes it undergoes as it folds to the native state. Kinetic data for 29 Im7 point mutants show that the intermediate contains three of the four helices found in the native structure, packed around a specific hydrophobic core. However, the intermediate contains many non-native interactions; as a result, hydrophobic interactions become disrupted in the rate-limiting transition state before the final helix docks onto the developing structure. The results of this study support a hierarchical mechanism of protein folding and explain why the misfolding of Im7 occurs. The data also demonstrate that non-native interactions can play a significant role in folding, even for small proteins with simple topologies.

Colicin endonucleases and the H-N-H family of homing enzymes share a common active site structural motif that has similarities to the active sites of a variety of other nucleases such as the non-specific endonuclease from Serratia and the sequence-specific His-Cys box homing enzyme I-PpoI. In contrast to these latter enzymes, however, it remains unclear how H-N-H enzymes cleave nucleic acid substrates. Here, we show that the H-N-H enzyme from colicin E9 (the E9 DNase) shares many of the same basic enzymological characteristics as sequence-specific H-N-H enzymes including a dependence for high concentrations of Mg2+ or Ca2+ with double-stranded substrates, a high pH optimum (pH 8-9) and inhibition by monovalent cations. We also show that this seemingly non-specific enzyme preferentially nicks double-stranded DNA at thymine bases producing 3'-hydroxy and 5'-phosphate termini, and that the enzyme does not cleave small substrates, such as dinucleotides or nucleotide analogues, which has implications for its mode of inhibition in bacteria by immunity proteins. The E9 DNase will also bind single-stranded DNA above a certain length and in a sequence-independent manner, with transition metals such as Ni2+ optimal for cleavage but Mg2+ a poor cofactor. Ironically, the H-N-H motif of the E9 DNase although resembling the zinc binding site of a metalloenzyme does not support zinc-mediated hydrolysis of any DNA substrate. Finally, we demonstrate that the E9 DNase also degrades RNA in the absence of metal ions. In the context of current structural information, our data show that the H-N-H motif is an adaptable catalytic centre able to hydrolyse nucleic acid by different mechanisms depending on the substrate and metal ion regime., (Copyright 2001 Academic Press.)

Immunity proteins are high affinity inhibitors of colicins--SOS-induced toxins released by bacteria during times of stress. Recent work has shown that nuclease-specific immunity proteins are exosite inhibitors, binding adjacent to the enzyme active site and inhibiting colicin activity indirectly. Unusually, their binding sites comprise a near contiguous sequence that lies N-terminal to active site sequences, raising the possibility that immunity proteins bind colicins co-translationally. Exosite binding accounts for the extensive sequence diversity seen at the interfaces of colicin-immunity protein complexes, which is not only a selective advantage to colicin-producing bacteria, but also represents a powerful model system for studying specificity in protein-protein recognition.

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins., (Copyright 2001 Academic Press.)

To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (k(UN)(H(2)O)) approximately 2000 s(-1) at pH 7.0, 25 degrees C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (beta(TS)0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)(Im9), Im2 (1-64)(Im9 )and Im2 (25-44)(Im9)) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)(Im9) and Im2 (56-64)(Im9) fold in two-state transitions via transition states that are significantly less compact (beta(TS) approximately 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (beta(TS) approximately 0.9). Surprisingly, Im2 (30-64)(Im9) populates a highly collapsed species (beta(I)=0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape., (Copyright 2001 Academic Press.)

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called sigmaR in response to changes in the cytoplasmic thiol-disulphide status, and the activity of sigmaR is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the anti-sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol-disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and sigmaR-dependent transcription, confirming that RsrA is a negative regulator of sigmaR and a key sensor of thiol-disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near-stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol-disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti-sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free sigmaR.

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.

The complex between the ribonuclease domain of the ribosome-inactivating colicin E3 and its protein inhibitor, the cognate immunity Im3, has been crystallized and preliminary X-ray characterization has been performed. Single crystals of the 1:1 complex were grown from hanging-drop vapour-diffusion experiments using 2-propanol as a precipitant. The space group is P3(1)21 or P3(2)21, with unit-cell parameters a = b = 93.7, c = 76.2 A. When cryocooled, these crystals diffract to a resolution of 2.4 A. A search for suitable conventional heavy-atom derivatives was unsuccessful and so Im3 mutants containing engineered cysteine or methionine residues have been produced for mercury soaks and selenomethionine-labelling experiments, respectively.

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.

Background: The cytotoxicity of most ribonuclease E colicins towards Escherichia coli arises from their ability to specifically cleave between bases 1493 and 1494 of 16S ribosomal RNA. This activity is carried by the C-terminal domain of the colicin, an activity which if left unneutralised would lead to destruction of the producing cell. To combat this the host E. coli cell produces an inhibitor protein, the immunity protein, which forms a complex with the ribonuclease domain effectively suppressing its activity., Results: We have solved the crystal structure of the cytotoxic domain of the ribonuclease colicin E3 in complex with its immunity protein, Im3. The structure of the ribonuclease domain, the first of its class, reveals a highly twisted central beta-sheet elaborated with a short N-terminal helix, the residues of which form a well-packed interface with the immunity protein., Conclusions: The structure of the ribonuclease domain of colicin E3 is novel and forms an interface with its inhibitor which is significantly different in character to that reported for the DNase colicin complexes with their immunity proteins. The structure also gives insight into the mode of action of this class of enzymatic colicins by allowing the identification of potentially catalytic residues. This in turn reveals that the inhibitor does not bind at the active site but rather at an adjacent site, leaving the catalytic centre exposed in a fashion similar to that observed for the DNase colicins. Thus, E. coli appears to have evolved similar methods for ensuring efficient inhibition of the potentially destructive effects of the two classes of enzymatic colicins.

Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme., (Copyright 1998 Academic Press.)

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3-84 were 0.76 +/- 0.14 A for the backbone atoms and 1.36 +/- 0.15 A for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 A, respectively, for residues 4-83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H-1H-13C NOESY-HSQC spectra with and without 13C decoupling. The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.

The Lit protease in Escherichia coli K-12 strains induces cell death in response to bacteriophage T4 infection by cleaving translation elongation factor (EF) Tu and shutting down translation. Suicide of the cell is timed to the appearance late in the maturation of the phage of a short peptide sequence in the major head protein, the Gol peptide, which activates proteolysis. In the present work we demonstrate that the Gol peptide binds specifically to domains II and III of EF-Tu, creating the unique substrate for the Lit protease, which then cleaves domain I, the guanine nucleotide binding domain. The conformation of EF-Tu is important for binding and Lit cleavage, because both are sensitive to the identity of the bound nucleotide, with GDP being preferred over GTP. We propose that association of the T4 coat protein with EF-Tu plays a role in phage head assembly but that this association marks infected cells for suicide when Lit is present. Based on these data and recent observations on human immunodeficiency virus type 1 maturation, we speculate that associations between host translation factors and coat proteins may be integral to viral assembly in both prokaryotes and eukaryotes.

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N-1H-15N HSQC-NOESY-HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 +/- 0.2 s(-1) at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 +/- 0.11 s(-1) at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 A radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.

SigR (sigma(R)) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic-producing, Gram-positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox-sensitive, sigma(R)-specific anti-sigma factor, RsrA, which binds sigma(R) and inhibits sigma(R)-directed transcription in vitro only under reducing conditions. Exposure to H(2)O(2) or to the thiol-specific oxidant diamide caused the dissociation of the sigma(R)-RsrA complex, thereby allowing sigma(R)-dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that sigma(R), RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol-disulfide redox balance.

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C. These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms. The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0. 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried). By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125). Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data. The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general., (Copyright 1999 Academic Press.)

The crystal structure of the cytotoxic endonuclease domain from the bacterial toxin colicin E9 in complex with its cognate immunity protein Im9 reveals that the inhibitor does not bind at the active site, the core of which comprises the HNH motif found in intron-encoded homing endonucleases, but rather at an adjacent position leaving the active site exposed yet unable to bind DNA because of steric and electrostatic clashes with incoming substrate. Although its mode of action is unorthodox, Im9 is a remarkably effective inhibitor since it folds within milliseconds and then associates with its target endonuclease at the rate of diffusion to form an inactive complex with sub-femtomolar binding affinity. This hyperefficient mechanism of inhibition could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active site.

We have crystallized and performed preliminary X-ray characterization of the complex between the DNAase domain of the E9 colicin and its cognate immunity protein Im9. The dissociation constant for this complex, Kd = 1 x 10(-16) M, reveals it to be one of the highest affinity protein-protein interactions known. Single crystals of the 1:1 complex were grown from microseeding experiments using PEG 4K as precipitant. The space group is P212121 with one molecule of complex in the asymmetric unit, and crystals contain approximately 43% solvent. These crystals are inherently non-isomorphous and so selenomethionine-derivatized protein has been prepared and crystals grown for MAD phasing experiments.

The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.

The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a Kd of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin-immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein-protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, using alanine mutagenesis of the putative endonuclease binding surface of Im2, compare the distribution of binding energies for conserved and nonconserved sites in both immunity proteins. This comparison highlights the conserved residues of both Im2 and Im9 as the major determinants of E9 DNase binding energy. Conversely, the nonconserved, specificity-determining residues only contribute to the E9 DNase binding energy in the cognate Im9 protein, while in the noncognate immunity protein Im2, they either destabilize the complex or do not contribute to the binding energy. This comparative alanine scan of two immunity proteins therefore supports the dual recognition mechanism of selectivity in colicin-immunity protein interactions and provides a basis for understanding specificity in other protein-protein interaction systems involving structurally conserved protein families.

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid 15N- and 13C/15N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C alpha and C beta chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D 1H-1H-15N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 degrees C with a forward rate constant of 1.61 +/- 0.5 s-1 and a backward rate constant of 1.05 +/- 0.5 s-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743-13750 and 13751-13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI. is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form. The possibility that cis-trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA [symbol: see text] NB interconversion to be identified, Pro65 and Pro73. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.

We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.

Immunity proteins inhibit colicins, protein toxins released by bacteria during times of environmental stress, by binding and inactivating their cytotoxic domains. This protects the producing organism as it attempts to kill off competing bacteria. The cytotoxic domains of related colicins share a high degree of sequence identity, as do their corresponding immunity proteins, yet specificity and affinity are also high, with little non-cognate biological cross-protection evident under physiological conditions. We review recent work on DNase-specific immunity proteins, which shows that, although both cognate and non-cognate proteins can bind a single toxin, their affinities can differ by as much as 12 orders of magnitude. We have termed this mode of binding dual recognition, because the DNase-binding surface of an immunity protein is made up of two components, one conserved and the other variable. The strength of the binding interaction is dominated by the conserved residues, while neighbouring variable residues control specificity. Similar dual recognition systems may exist in other biological contexts, particularly where a protein must discriminate the right binding partner from numerous, structurally homologous alternatives.

The endonuclease group of E colicins are a family of bacterial toxins whose cytotoxic activity in a producing host is inactivated by a specific immunity protein. The DNase of colicin E9 can be bound and inhibited by both cognate and noncognate immunity proteins, the dissociation constants for which span a range of 12-orders of magnitude. DNase binding specificity of the immunity proteins is governed primarily by helix II, the sequence of which is variable in this family of proteins. Heteronuclear NMR experiments have identified helix III along with helix II as the likely DNase binding site, although other regions of Im9 also showed perturbations on binding the E9 DNase. In the present work, we have used the NMR experiments as a guide for alanine scanning mutagenesis of Im9. Our data show that helices II and III of Im9 are indeed the DNase binding site and in addition quantitate the relative binding energy associated with each helix. We find that the conserved residues of helix III make the largest relative contribution toward E9 DNase binding. In conjunction with previous studies, the data suggest that specificity in the colicin-immunity system is governed by a dual recognition mechanism in which highly stabilizing interactions emanating from the conserved regions of an immunity protein act as the binding site anchor and these are modulated by interactions from neighboring, nonconserved amino acid residues. This modulation is likely to take the form of both favorable and unfavorable interactions, the balance of which define the specificity of the protein-protein interaction. The generality of such a dual recognition mechanism in other systems is also discussed.

E colicins are plasmid-coded, protein antibiotics which bind to the BtuB outer membrane receptor of Escherichia coli cells and are then translocated either to the outer surface of the cytoplasmic membrane in the case of the pore-forming colicin E1, or to the cytoplasm in the case of the enzymic colicins E2-E9. Translocation has been proposed to be dependent on a putative TolA box; a pentapeptide (DGSGW) located in the N-terminal 39 residues of several Tol-dependent colicins. In this study, site-directed mutagenesis was used to change each of the residues of the putative TolA box of colicin E9 to alanines. In the case of the two glycine residues, the resulting mutant proteins were indistinguishable from the native colicin E9 protein in a biological assay; whereas the other three residues when mutated to alanines resulted in a complete loss of biological activity. Mutagenesis of two serine residues flanking the putative TolA box, Ser34 and Ser40, to alanines did not abolish the biological activity of the mutant colicin E9, although the zones of growth inhibition were hazy and slow to form. The size of the zone of inhibition was significantly smaller than the control in the case of the Ser40Ala mutant. The ColE9/Im9 complex was isolated from the three biologically inactive TolA box alanine mutants. In competition assays all three mutant protein complexes were capable of protecting sensitive E. coli cells against killing by the native ColE9/Im9 complex. On removal of the Im9 protein from the three mutant ColE9/Im9 complexes, all three mutant colicins exhibited DNase activity. These results confirm the importance of the putative TolA box in the biological activity of colicin E9, and suggest that the TolA box has a role in the translocation of colicin E9.

1H-15N NMR studies, in conjunction with mutagenesis experiments, have been used to delineate the DNase-binding surface of the colicin E9 inhibitor protein Im9 (where Im stands for immunity protein). Complexes were formed between the 15 kDa unlabelled E9 DNase domain and the 9.5 kDa Im9 protein uniformly labelled with 15N. Approx. 90% of the amide resonances of the bound Im9 were assigned and spectral parameters obtained from 1H-15N heteronuclear single quantum coherence (HSQC) spectra were compared with those for the free Im9 assigned previously. Many of the amide resonances were shifted on complex formation, some by more than 2 p.p.m. in the 15N dimension and more than 0.5 p.p.m. in the 1H dimension. Most of the strongly shifted amides are located on the surfaces of two of the four helices, helix II and helix III. Whereas helix II had already been identified through genetic and biochemical investigations as an important determinant of biological specificity, helix III had not previously been implicated in binding to the DNase. To test the robustness of the NMR-delineated DNase-binding site, a selection of Im9 alanine mutants were constructed and their dissociation rate constants from E9 DNase-immunity protein complexes quantified by radioactive subunit exchange kinetics. Their off-rates correlated well with the NMR perturbation analysis; for example, residues that were highly perturbed in HSQC experiments, such as residues 34 (helix II) and 54 (helix III), had a marked effect on the DNase-immunity protein dissociation rate when replaced by alanine. The NMR and mutagenesis data are consistent with a DNase-binding region on Im9 composed of invariant residues in helix III and variable residues in helix II. The relationship of this binding site model to the wide range of affinities (Kd values in the range 10(-4) to 10(-16)M) that have been measured for cognate and non-cognate colicin DNase-immunity protein interactions is discussed.

A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coli aroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

We have investigated the involvement of metal ions and conformational changes in the elimination reaction catalysed by type II dehydroquinase from Aspergillus nidulans. Mechanistic comparisons between dehydroquinases and aldolases raised the possibility that, by analogy with type II aldolases, type II dehydroquinases may require bivalent metal ions for activity. This hypothesis was tested by a combination of metal analysis, effects of metal chelators and denaturation/renaturation experiments, all of which failed to show any evidence that type II dehydroquinases are metal-dependent dehydratases. Analysis of native and refolded enzyme by electron microscopy showed that the dodecameric type II enzyme from A. nidulans adopts a ring-like structure similar to that of glutamine synthase, suggesting an arrangement of two hexameric rings stacked on top of one another. Evidence for a ligand-induced conformational change came from both chemical modification and proteolysis experiments. Inactivation data with the arginine-specific reagent phenylglyoxal indicated that, at pH 7.5, two arginine residues are modified: one modification displays affinity-labelling kinetics and has a 1:1 stoichiometry, while the other displays simple bimolecular kinetics and a stoichiometry of 2:1. The labelling at the affinity site is markedly enhanced by the addition of ligand, implying that this active-site residue is further exposed to modification by phenylglyoxal as a result of a ligand-induced conformational change. A combination of proteolysis and electrospray MS experiments identified the site of affinity labelling as Arg-19. The highly conserved N-terminal region encompassing Arg-19 of type II dehydroquinase was found to be particularly susceptible to proteolytic cleavage Limited digestion with proteinase K inactivates the enzyme, although the type II oligomeric structure is retained, and ligand binding partially protects against this inactivation.

We have used random mutagenesis to identify putative active-site residues in the C-terminal cytotoxic endonuclease domain of the bacterial toxin colicin E9. Six single-site mutations in the DNase domain were isolated which destroyed the toxic action of the colicin. DNA sequencing identified the mutations as Gly460Asp, Arg544Gly, Glu548Gly, Thr571Ile, His575Tyr and His579Tyr. All six wild-type residues are highly conserved in the DNase domains of both the E group colicins and the closely related pyocins. Site-directed mutagenesis was then used to substitute the wild-type amino acid residue at each of these positions for an alanine residue in order to distinguish important from unimportant sites. Two of the six alanine-mutant colicins (Gly460Ala and His579Ala) exhibited significant in vivo activity, unlike the original mutation of these residues, and were therefore not characterised further. The Thr571Ala mutant colicin, although not inactive, was significantly less active than the control. The other three alanine mutants (Arg544Ala, Glu548Ala and His575Ala remained completely inactive in the in vivo tests. Each 15 kDa alanine-mutant DNase domain was overexpressed and purified using a tandem-expression strategy which relies on the enzyme being able to bind to the natural inhibitor, Im9. Tryptophan emission spectra of the alanine mutants showed significant alterations in the emission maxima of all but the His575Ala mutant, suggesting changes in the tertiary structure of these mutant proteins. Activity measurements, using the spectrophotometric Kunitz assay, indicated that the Thr571Ala mutant was partially active as an endonuclease but the remaining alanine mutants were all completely inactive. All four mutant proteins, however, retained their ability to bind DNA in a gel shift assay, suggesting the mutations affect catalytic rather than substrate-binding residues. Searching the sequence databases for possible homology to other DNA-binding proteins revealed a significant match between residues 464 to 487 of the E9 DNase domain and helix IV of the POU domain of eukaryotic transcription factors.

The 86-amino acid colicin E9 immunity protein (Im9), which inhibits the DNase activity of colicin E9, has been overexpressed in Escherichia coli and isotopically enriched with 15N and 13C. Using the 3D CBCANH and CBCA(CO)NH experiments, we have almost completely assigned the backbone 13C resonances and extended previously reported 15N/1H backbone assignments [Osborne et al. (1994), Biochemistry 33, 12347-12355]. Side chain assignments for almost all residues were made using the 3D 13C HCCH-TOCSY experiment allied to previous 1H assignments. Sixty solution structures of Im9 were determined using the DIANA program on the basis of 1210 distance restraints and 56 dihedral angle restraints. The 30 lowest-energy structures were then subjected to a slow-cooling simulated annealing protocol using XPLOR and the 21 lowest-energy structures, satisfying the geometric restraints chosen for further analysis. The Im9 structure is well-defined except for the termini and two solvent-exposed loops between residues 28-32 and 57-64. The average RMSD about the average structure of residues 4-84 was 0.94 A for all heavy atoms and 0.53 A for backbone C alpha, C = O, and N atoms. The Im9 fold is novel and can be considered a distorted antiparallel four-helix bundle, in which the third helix is rather short, being terminated close to its N-terminal end by a proline at its C-terminus. The structure fits in well with available kinetic and biochemical data concerning the interaction between Im9 and its target DNase. Important residues of Im9 that govern specificity are located on the molecular surface in a region rich in negatively charged groups, consistent with the proposed electrostatically steered association [Wallis et al. (1995a), Biochemistry 34, 13743-13750].

The in vivo and in vitro cross-binding of the colicin endonuclease-specific immunity proteins toward the DNase domain of colicin E9 is described. In vivo cross-protection was tested by toxin plate assays in which bacterial cells overexpressing each immunity (Im2, Im7, Im8, and Im9) were challenged with the ColE9 toxin. Im9, the cognate immunity protein, renders cells completely resistant toward very high concentrations of the toxin (> 1 mg/mL), whereas the noncognate immunities display a spectrum of weaker cross-reactivities (< 0.01 mg/mL). The order of biological protection in this assay was Im9 >> Im2 > Im8, with Im7 providing no colicin E9 resistance. In vitro binding between the immunity proteins and the E9 DNase was analyzed by determining the dissociation constants for E9 DNase-Im protein complexes at pH 7.0 in the presence of 200 mM salt and at 25 degrees C. Stopped-flow fluorescence experiments suggest that both Im2 and Im8 associate with the E9 DNase by a two-step mechanism, in which the rate constants for both the bimolecular association (k1 = approximately 6 x 10(7) M-1 s-1) and the subsequent conformational change (k2 + k-2 = 4-5 s-1) are very similar to Im9 binding under the same conditions. Fluorescence chase experiments defined the dissociation rate constants for Im2 and Im8. The estimated values are 10(6)- and 10(8)-fold, respectively, faster than the off-rate for the Im9 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a nonspecific DNase housed in the C-terminus of the protein. A kinetic and thermodynamic analysis of complex formation for both the holotoxin and the isolated DNase domain with the cytoplasmic inhibitor of this enzyme, the immunity protein Im9, is presented. The dissociation constant for each complex was calculated from the ratio of the association and dissociation rate constants. Association was monitored by stopped-flow fluorescence and comprises at least two steps for both complexes, an initial fluorescence enhancement followed by a fluorescence quench. The data are consistent with a two-step binding mechanism in which the rate of formation of an encounter complex (k1) is rate determining and essentially diffusion controlled (4.0 x 10(9) M-1 s-1 for colicin E9) in buffer of low ionic strength. This encounter complex then rearranges to the final stable complex. Sequential stopped-flow experiments using 5-hydroxy-L-tryptophan labeled DNase domain support the two-step mechanism and further show that the rate of encounter complex rearrangement is significantly faster than its dissociation. The overall rate of dissociation of the colicin E9-Im9 complex (k(off)) was determined by radioactive subunit exchange to be 3.7 x 10(-7) s-1. Thus, the Kd for the complex (k(off)/k1) is 9.3 x 10(-17) M, which corresponds to a change in free energy on binding of -21.9 kcal mol-1 at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

1H-1H, 1H-15N, and 1H-1H-15N multidimensional NMR spectroscopic studies of the 86 amino acid protein that provides immunity against the DNase action of colicin E9 are reported. Through a combination of 2D NOESY and TOCSY and 3D TOCSY-HMQC, NOESY-HMQC, and HMQC-NOESY-HMQC experiments, almost complete 1H NMR and backbone 15N NMR assignments have been obtained, and the secondary structure of the protein has been partially elucidated. Approximately 50% of the protein forms three helices. The specificity determining region of the DNase immunity protein, identified from previously reported biochemical studies to include residues 32-40, is helical, indicating that the protein-protein interaction involves residues from at least one helix.

We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.

The specificity-determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16-43 of the 86-amino-acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity-determining residues. Using site-directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity-determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low-level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity-determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5'-dithiobis(2-nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti-DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity-determining region.