Calorimetric dissection of colicin DNase--immunity protein complex specificity

We explore the thermodynamic strategies used to achieve specific, high-affinity binding within a family of conserved protein-protein complexes. Protein-protein interactions are often stabilized by a conserved interfacial hotspot that serves as the anchor for the complex, with neighboring variable residues providing specificity. A key question for such complexes is the thermodynamic basis for specificity given the dominance of the hotspot. We address this question using, as our model, colicin endonuclease (DNase)-immunity (Im) protein complexes. In this system, cognate and noncognate complexes alike share the same mechanism of association and binding hotspot, but cognate complexes (K(d) approximately 10(-)(14) M) are orders of magnitude more stable than noncognate complexes (10(6)-10(10)-fold discrimination), largely because of a much slower rate of dissociation. Using isothermal titration calorimetry (ITC), we investigated the changes in enthalpy (DeltaH), entropy (-TDeltaS), and heat capacity (DeltaC(p)) accompanying binding of each Im protein (Im2, Im7, Im8, and Im9) to the DNase domains of colicins E2, E7, E8, and E9, in the context of both cognate and noncognate complexes. The data show that specific binding to the E2, E7, and E8 DNases is enthalpically driven but entropically driven for the E9 DNase. Analysis of DeltaC(p), a measure of the change in structural fluctuation upon complexation, indicates that E2, E7, and E8 DNase specificity is coupled to structural changes within cognate complexes that are consistent with a reduction in the conformational dynamics of these complexes. In contrast, E9 DNase specificity appears coupled to the exclusion of water molecules, consistent with the nonpolar nature of the interface of this complex. The work highlights that although protein-protein interactions may be centered on conserved structural epitopes the thermodynamic mechanism underpinning binding specificity can vary considerably.     

Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes

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.     

Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases

Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K_d values that vary between 10^− 4 and 10^− 14 M, characteristics that are explained by a ‘dual-recognition’ mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential ΔΔGs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental ΔΔG data, particularly for interactions not mediated by interfacial water molecules. ΔΔG predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites.     

Structure of the ultra-high-affinity colicin E2 DNase--Im2 complex

How proteins achieve high-affinity binding to a specific protein partner while simultaneously excluding all others is a major biological problem that has important implications for protein design. We report the crystal structure of the ultra-high-affinity protein-protein complex between the endonuclease domain of colicin E2 and its cognate immunity (Im) protein, Im2 (K(d)～10(-)(15)?M), which, by comparison to previous structural and biophysical data, provides unprecedented insight into how high affinity and selectivity are achieved in this model family of protein complexes. Our study pinpoints the role of structured water molecules in conjoining hotspot residues that govern stability with residues that control selectivity. A key finding is that a single residue, which in a noncognate context massively destabilizes the complex through frustration, does not participate in specificity directly but rather acts as an organizing center for a multitude of specificity interactions across the interface, many of which are water mediated.     

Multistep binding of transition metals to the H-N-H endonuclease toxin colicin E9

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.     

Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin.

The directed mutagenesis study of the Im7 protein of colicin E7 revealed that three residues, D31, D35, and E39, located in the loop 1 and helix 2 regions of the protein were critical for initiating the complex formation with its cognate colicin E7. Interestingly, the importance of these three critical residues in conferring specific immunity to its own colicin was exhibited in a hierarchical order, respectively. Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins. Most likely the three residues of the DNase-type immunity proteins are critical for initiating the unique protein-protein interactions with their cognate colicin. In addition, replacement of the helix 2 of Im7 by the corresponding region of Im8 produced a phenotype of the mutant protein very similar to that of Im8. This result suggests that the DNase-type Im proteins indeed share a "homologous-structural framework" and evolution of the Im proteins may be engendered by minor amino acid changes in this specific immunity-determining region without causing structural alteration of the proteins.     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.     

Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry

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.     

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.     

Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.     

A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells

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.     

Immunity protein protects colicin E2 from OmpT protease

The endonuclease colicin E2 (ColE2), a bacteriocidal protein, and the associated cognate immunity protein (Im2) are released from producing Escherichia coli cells. ColE2 interaction with the target cell outer membrane BtuB protein and Tol import machinery allows the dissociation of Im2 from its colicin at the outer membrane surface. Here, we use in vivo approaches to show that a small amount of ColE2-Im2 protein complex bound to sensitive cells is susceptible to proteolytic cleavage by the outer membrane protease, OmpT. The presence of BtuB is required for ColE-Im2 cleavage by OmpT. The amount of colicin cleaved by OmpT is greatly enhanced when ColE2 is dissociated from Im2. We further demonstrate that OmpT cleaves the C-terminal DNase domain of the toxin. As expected, strains that over-produce OmpT are less susceptible to infection by ColE2 than by ColE2-Im2. Our findings reveal an additional function for the immunity protein beside protection of producing cells against their own colicin in the cytoplasm. Im2 protects ColE2 against OmpT-mediated proteolytic attack.     

Destabilization of the colicin E9 Endonuclease domain by interaction with negatively charged phospholipids: implications for colicin translocation into bacteria

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lema?tre, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (lambda(max)) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in lambda(max), from 354 to 342 nm. Changes in lambda(max) were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.     

Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery

In vitro compartmentalization (IVC) uses water-in-oil emulsions to create artificial cell-like compartments in which genes can be individually transcribed and translated. Here, we present a new application of IVC for the selection of DNA-nuclease inhibitors. We developed a nano-droplets delivery system that allows the transport of various solutes, including metal ions, into the emulsion droplets. This transport mechanism was used to regulate the activity of colicin nucleases that were co-compartmentalized with the genes, so that the nucleases were activated by nickel or cobalt ions only after the potential inhibitor genes have been translated. Thus, genes encoding nuclease inhibitors survived the digestion and were subsequently amplified and isolated. Selection is therefore directly for inhibition, and not for binding of the nuclease. The stringency of selection can be easily modulated to give high enrichments (100-500-fold) and recoveries. We demonstrated its utility by selecting libraries of the gene encoding the cognate inhibitor of colicin E9 (immunity protein 9, or Im9) for inhibition of another colicin (ColE7). The in vitro evolved inhibitors show significant inhibition of ColE7 both in vitro and in vivo. These Im9 variants carry mutations into residues that determine the selectivity of the natural counterpart (Im7) while completely retaining the residues that are conserved throughout the family of immunity protein inhibitors. The in vitro evolution process confirms earlier hypotheses regarding the "dual recognition" binding mechanism and the way in which new colicin-immunity pairs diverged from existing ones.     

The kinetic basis for dual recognition in colicin endonuclease-immunity protein complexes

The antibacterial activity of E colicin endonucleases (DNases) is counteracted by the binding of immunity proteins; the affinities of cognate and non-cognate complexes differing by up to ten orders of magnitude. Here, we address the mechanism of complex formation using a combination of protein engineering, pre-steady-state kinetics and isothermal titration calorimetry, in order to understand the underlying basis for specificity. Contrary to previous work, we show that a pre-equilibrium mechanism does not explain the binding kinetics. Instead, the data are best explained by a modified induced-fit mechanism where cognate and non-cognate complexes alike form a non-specific, conformationally dynamic encounter complex, most likely centred on conserved interactions at the interface. The dynamics appear to be an intrinsic property of the encounter complex where the proteins move relative to one another, thereby sampling different conformations rather than being "induced" by binding. This allows optimal alignment of interface specificity sites, without producing energetically costly conformational changes, essential for high-affinity binding. Importantly, specificity is achieved without slowing the rate of association, an important requirement for rapid inhibition of the colicin in the producing bacterial cell. A rigid-body rotation model is also consistent with the observation that specificity contacts in colicin-immunity protein complexes can involve different regions of the interface. Such a kinetic discrimination mechanism explains the ability of DNase-specific immunity proteins to display dual recognition specificity, wherein they are broadly cross-reactive yet are highly specific, achieving femtomolar binding affinities in complexes with their cognate DNases.     

Site-specific labeling of proteins with cyclen-bound transition metal ions

A general procedure for site-specific and reversible labeling of proteins with transition metal ions is described. The method is based on the use of the novel ligand 1-(2-thioethyl)-1,4,7,10-tetraazacyclododecane (TETAC), which specifically and readily reacts with thiol groups. Synthesis of TETAC from 1,4,7,10-tetraazacyclododecane (cyclen) and ethylene disulfide yielded a mixture of products, including TETAC and its oxidized disulfide in 56.4% yield. The procedure for labeling proteins with TETAC is straightforward and led to separation of the TETAC-containing product mixture through gel-filtration chromatography. The resulting protein-TETAC adducts were shown to contain a single TETAC group which bound transition metal ions. Protein-TETACCu²⁺ had a UV-Vis spectrum similar to that of Cu²⁺(cyclen) while the protein-TETACCo²⁺ complex had a different spectrum to that of the cobalt-containing cyclen. This is because attachment to the protein prevented the Co²⁺-containing TETAC from dimerising and binding O₂, which the cobalt-containing cyclen is able to do. The proteins used to develop this labeling procedure were the DNase domain of colicin E9 and its inhibitor protein Im9. Unlike Im9, the DNase does not contain a cysteine residue but the Ser30Cys variant of the DNase was prepared by site-directed mutagenesis. Both Im9 and the Ser30Cys DNase were modified with TETAC and the modifications shown to be structurally and functionally benign through NMR spectroscopy of the modified Im9 and fluorescence spectroscopy binding assays in which DNase-Im9 complexes were formed. The simplicity of the method, and its general application to any protein through the introduction of cysteine by site-directed mutagenesis, suggests it will be of wide use in protein chemistry applications.     

Release of immunity protein requires functional endonuclease colicin import machinery

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.     

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

Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (K_d≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 10⁶-fold, to a timescale (lifetime ≈10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods.     

Computational design of a new hydrogen bond network and at least a 300-fold specificity switch at a protein-protein interface

The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.     

Molecular basis of inhibition of the ribonuclease activity in colicin E5 by its cognate immunity protein

Colicin E5 is a tRNA-specific ribonuclease that recognizes and cleaves four tRNAs in Escherichia coli that contain the hypermodified nucleoside queuosine (Q) at the wobble position. Cells that produce colicin E5 also synthesize the cognate immunity protein (Im5) that rapidly and tightly associates with colicin E5 to prevent it from cleaving its own tRNAs to avoid suicide. We report here the crystal structure of Im5 in a complex with the activity domain of colicin E5 (E5-CRD) at 1.15A resolution. The structure reveals an extruded domain from Im5 that docks into the recessed RNA binding cleft in E5-CRD, resulting in extensive interactions between the two proteins. The interactions are primarily hydrophilic, with an interface that contains complementary surface charges between the two proteins. Detailed interactions in three separate regions of the interface account for specific recognition of colicin E5 by Im5. Furthermore, single-site mutational studies of Im5 confirmed the important role of particular residues in recognition and binding of colicin E5. Structural comparison of the complex reported here with E5-CRD alone, as well as with a docking model of RNA-E5-CRD, indicates that Im5 achieves its inhibition by physically blocking the cleft in colicin E5 that engages the RNA substrate.