The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins (original) (raw)
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Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB
Journal of Molecular Biology, 2002
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 ðt m ¼ 200 msÞ: 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 spinspin 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.
Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box
Journal of Bacteriology, 2005
The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.
Biochemistry, 2002
Various macromolecules such as bacteriotoxins and phage DNA parasitize some envelope proteins of Escherichia coli to infect the bacteria. A two-step import mechanism involves the primary interaction with an outer membrane receptor or with a pilus followed by the translocation across the outer membrane. However, this second step is poorly understood. It was shown that the TolA, TolQ, and TolR proteins play a critical role in the translocation of group A colicins and filamentous bacteriophage minor coat proteins (g3p). Translocation of these proteins requires the interaction of their N-terminal domain with the C-terminal domain of TolA (TolAIII). In this work, short soluble TolAIII domains were overproduced in the cytoplasm and in the periplasm of E. coli. In TolAIII, the two cysteine residues were found to be reduced in the cytoplasmic form and oxidized in the periplasmic form. The interaction of TolAIII with the N-terminal domain of colicin A (ATh) is observed in the presence and in the absence of the disulfide bridge. The complex formation of TolAIII and ATh was found to be independent of the ionic strength. An NMR study of TolAIII, both free and bound, shows a significant structural change when interacting with ATh, in the presence or absence of the disulfide bridge. In contrast, such a structural modification was not observed when TolAIII interacts with g3p N1. These results suggest that bacteriotoxins and Ff bacteriophages parasitize E. coli using different interactions between TolA and the translocation domain of the colicin and g3p protein, respectively.
The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins
Biochimie, 2002
The Tol proteins of Escherichia coli are involved in outer membrane stability. They are also required for the uptake of the group A colicins and the translocation of filamentous phage DNA into the cytoplasm. The tol-pal genes constitute two operons in the E. coli genome, orf1tolQRA and tolBpalorf2. The TolQ TolR TolA proteins form a complex in the cytoplasmic membrane, while TolB and Pal interact near the outer membrane. Most of the amino acid residues of TolA, TolB, TolR and Pal are localized in the periplasm. Recent advances in the knowledge of interactions of Tol-Pal proteins with other envelope components, or with group A colicins, are presented, together with current hypotheses about the role of the Tol proteins in outer membrane stability.
Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9
Journal of Biomolecular NMR, 2000
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.
TURKISH JOURNAL OF BIOLOGY, 2014
Introduction Colicins are plasmid-encoded bactericidal proteins produced by immune Escherichia coli that are active against sensitive E. coli and its closely related cells. Their toxic activities are of various types: some colicins form ion channels in the cytoplasmic membrane of sensitive cells, while others act as nucleases that degrade DNA or 16S RNA in the cytoplasm. One colicin, colicin M, inhibits the biosynthesis of murein (Lakey et al., 1994). Their toxic activities against target cells are known to occur in 3 distinct stages (Figure 1). First is receptor recognition and binding, where colicins bind to a specific receptor at the cell surface. Second is the translocation step, where colicins cross both the outer membrane and the periplasm to reach their cellular target (Lakey et al., 1994; Lazdunski, 1995). The final stage is the killing action, where colicins exert their lethal effects by forming a pore in the cytoplasmic membrane (Lazdunski, 1995), by DNAse or RNAse activity, or by inhibiting murein biosynthesis in the cytoplasm (Lakey et al., 1994). Colicins have 3 linearly organized functional domains, each domain implicated in a specific stage of the colicin activity. As shown in Figure 1, the central domain (R-domain) is responsible for the receptor-binding activity. The N-terminal domain (T-domain) is involved in translocation. The C-terminal domain (P-domain) carries out the lethal activity; this domain either forms a voltagegated pore in the cytoplasmic membrane or digests nucleic acids in the cytoplasm (Raggett et al., 1998; Vetter et al., 1998; Papadokus et al., 2011). Colicin N is a group A (Tol-dependent) pore-forming colicin whose target receptor is the E. coli outer membrane protein OmpF (Pugsley, 1987). It is composed of a largely unstructured T-domain linked by a glycine-rich sequence to a central R-domain containing a 6-stranded β-sheet structure. The R-domain is connected to the P-domain (a 10 α-helical structure) by means of a 65 Å α-helix (El-Kouhen et al., 1993; Gokce et al., 2000). While receptor binding and pore formation have been extensively studied, much remains unknown about the translocation step. Colicin N translocation requires 3 members of the Tol locus: TolA, TolQ, and TolR (Webster, 1991). TolQ and TolR are integral membrane proteins of the E. coli inner membrane and there is no evidence that they reach across Abstract: Colicin N is a bacterial toxin that kills Escherichia coli and related cells. Its mode of action is of interest in protein import and toxicology. Colicin N translocates across the E. coli outer membrane and periplasm by interacting with several receptors. The translocation process involves the interaction of the colicin N with the outer membrane porin OmpF and subsequently with the integral membrane protein TolA. The N-terminal domain of colicin N is involved in the import process. TolA consists of 3 domains. The N-terminal domain of colicin N interacts with the C-terminal domain of TolA at later stages of the translocation process. Our aim was to produce a large quantity of colicin N T-domains for spectroscopic and crystallization studies. These both require a correctly folded and stable protein. Here we present an expression of the complex between the N-terminal domain of colicin N and the C-terminal domain of TolA obtained by fusing these 2 domains with a flexible linker. Circular dichroism spectroscopy studies indicated that unstructured bacterial toxin colicin N T-domains changed to an ordered state upon binding to the C-terminal domain of TolA; this fusion protein has a secondary and tertiary structure.
Proceedings of the National Academy of Sciences, 2006
The natively disordered N-terminal 83-aa translocation (T) domain of E group nuclease colicins recruits OmpF to a colicin-receptor complex in the outer membrane (OM) as well as TolB in the periplasm of Escherichia coli, the latter triggering translocation of the toxin across the OM. We have identified the 16-residue TolB binding epitope in the natively disordered T-domain of the nuclease colicin E9 (ColE9) and solved the crystal structure of the complex. ColE9 folds into a distorted hairpin within a canyon of the six-bladed beta-propeller of TolB, using two tryptophans to bolt the toxin to the canyon floor and numerous intramolecular hydrogen bonds to stabilize the bound conformation. This mode of binding enables colicin side chains to hydrogen-bond TolB residues in and around the channel that runs through the beta-propeller and that constitutes the binding site of peptidoglycan-associated lipoprotein (Pal). Pal is a globular binding partner of TolB, and their association is known to be important for OM integrity. The structure is therefore consistent with translocation models wherein the colicin disrupts the TolB-Pal complex causing local instability of the OM as a prelude to toxin import. Intriguingly, Ca(2+) ions, which bind within the beta-propeller channel and switch the surface electrostatics from negative to positive, are needed for the negatively charged T-domain to bind TolB with an affinity equivalent to that of Pal and competitively displace it. Our study demonstrates that natively disordered proteins can compete with globular proteins for binding to folded scaffolds but that this can require cofactors such as metal ions to offset unfavorable interactions.
Journal of Molecular Biology, 2000
Colicins translocate across the Escherichia coli outer membrane and periplasm by interacting with several receptors. After ®rst binding to the outer membrane surface receptors via their central region, they interact with TolA or TonB proteins via their N-terminal region. Colicin N residues critical to TolA binding have been discovered, but the full extent of any colicin TolA site is unknown. We present, for the ®rst time, a fully mapped TolA binding site for a colicin. It was determined through the use of alanine-scanning mutants, glutathione S-transferase fusion peptides and Biacore/¯uorescence binding studies. The minimal TolA binding region is 27 residues and of similar size to the TolA binding region of bacteriophage g3p-D1 protein. Stopped-¯ow kinetic studies show that the binding to TolA follows slow association kinetics. The role of other E. coli Tol proteins in colicin translocation was also investigated. Isothermal titration microcalorimetry (ITC) and in vivo studies conclusively show that colicin N translocation does not require the presence of TolB. ITC also demonstrated colicin A interaction with TolB, and that colicin A in its native state does not interact with TolAII-III. Colicin N does not bind TolR-II. The TolA protein is shown to be unsuitable for direct immobilisation in Biacore analysis.
Journal of Biological Chemistry, 1994
Colicin A is a bacterial toxin which forms channels in the cytoplasmic membrane of Escherichia coli. Its translocation through the envelope requires the participation of bacterial proteins encoded by the tole,-R,-A, and-B genes. Overproduction of the To1 proteins decreased the time needed for colicin A translocation and increased the number of channels formed in vivo. Cells overproducing radioactively labeled To1 proteins and containing or not colicin A were fractionated. The To1 proteins were mainly recovered in the inner membrane and in the contact sites between the two membranes. The presence of colicin A increased the specific radioactivity of the To1 proteins in the contact sites. Our data suggest that the To1 proteins form a complex of definite stoichiometry in the membranes and that colicin A is associated to this complex upon channel formation. We discuss the possibility that the channel activity determined in vivo is due to the colicin A-To1 proteins complex. Colicin A is a bacterial toxin (molecular mass = 63 kDa) produced by and active against Escherichia coli. Its COOHterminal domain carries the channel activity, the central domain is involved in the recognition of the receptor and the NHz-terminal domain is required for its translocation through the envelope (for reviews, see Lazdunski et al. (1988) and Pattus et al. (1990)). The channel activity of colicin A was first inferred from in vitro experiments (Pattus et al., 1983; Collarini et al., 1987). Recently, by analyzing the efflux of cytoplasmic potassium induced by colicin A, we have shown that the toxin forms voltage-gated channels in the cytoplasmic membrane of E. coli (Bourdineaud et al., 1990; reviewed in Letellier (1992)). The import of colicin A to the E. coli inner membrane is a multistep process; colicin A first binds to the outer membrane proteins BtuB and OmpF (Cavard and Lazdunski, 1981) and is then translocated through the envelope. This translocation requires the participation of bacterial proteins encoded by the to1
Molecular Microbiology, 2001
Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain. Both interactions are required for colicin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N-terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N-terminal domain and appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A colicin translocation.