Onkar Sharma

Crystal structures of the OmpF porin: function in a colicin translocon

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 Å OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co‐crystallization of OmpF with the unfolded 83 residue glycine‐rich N‐terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 Å structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly‐glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno‐extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.

Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon.

The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-Å crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg452 at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.

Conformational switching of the diphtheria toxin T domain.

The diphtheria toxin T domain translocates the catalytic C domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited wild-type-like activity in channel formation, N-terminus translocation, and cytotoxicity assays. Moreover, membrane permeabilization caused by the H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

Genome‐wide screens: novel mechanisms in colicin import and cytotoxicity

Only two new genes (fkpA and lepB) have been identified to be required for colicin cytotoxicity in the last 25 years. Genome‐wide screening using the ‘Keio collection’ to test sensitivity to colicins (col) A, B, D, E1, E2, E3, E7 and N from groups A and B, allowed identification of novel genes affecting cytotoxicity and provided new information on mechanisms of action. The requirement of lipopolysaccharide for colN cytotoxicity resides specifically in the lipopolysaccharide inner‐core and first glucose. ColA cytotoxicity is dependent on gmhB and rffT genes, which function in the biosynthesis of lipopolysaccharide and enterobacterial common antigen. Of the tol genes that function in the cytoplasmic membrane translocon, colE1 requires tolA and tolR but not tolQ for activity. Peptidoglycan‐associated lipoprotein, which interacts with the Tol network, is not required for cytotoxicity of group A colicins. Except for TolQRA, no cytoplasmic membrane protein is essential for cytotoxicity of group A colicins, implying that TolQRA provides the sole pathway for their insertion into/through the cytoplasmic membrane. The periplasmic protease that cleaves between the receptor and catalytic domains of colE7 was not identified, implying either that the responsible gene is essential for cell viability, or that more than one gene product has the necessary proteolysis function.

Chemical Dissection of Protein Translocation through the Anthrax Toxin Pore

Anthrax lethal toxin exemplifies one among many systems evolved by pathogenic bacteria for transporting proteins across membranes to the cytosol of mammalian cells.[1] The transported proteins so called effector proteins are enzymes that modify intracellular substrates, perturbing mammalian metabolism in ways that benefit the bacteria at the expense of the host. Anthrax lethal toxin is an ensemble of two large soluble proteins: the Lethal Factor (LF, 90 kDa), a zinc protease,[2] and Protective Antigen (PA; 83 kDa), a receptor-binding/pore-forming protein.[1] PA binds to receptors[3] on host cells and is cleaved by a furin-family protease[4] to an active 63 kDa form (PA63),[5] which self-assembles into ring-shaped heptameric[6] and octameric[7] oligomers, termed prepores. The prepores bind LF, forming complexes that are then endocytosed and delivered to the endosome. There, acidification induces the prepore moieties to undergo conformational rearrangement to membrane-spanning pores.[1] The pores then transport bound LF across the membrane to the cytosol, where it inactivates selected target proteins.[8] Edema Factor (EF), the enzymatic moiety of anthrax edema toxin,[9] is transported to the cytosol by a similar mechanism.[1]

Mobility of BtuB and OmpF in the Escherichia coli Outer Membrane: Implications for Dynamic Formation of a Translocon Complex

Diffusion of two Escherichia coli outer membrane proteins-the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages-was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05±0.01 μm2/s and 0.10±0.02 μm2/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27±0.06 μm2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006±0.002 μm2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

The colicin E3 outer membrane translocon: immunity protein release allows interaction of the cytotoxic domain with OmpF porin.

The crystal structure previously obtained for the complex of BtuB and the receptor binding domain of colicin E3 forms a basis for further analysis of the mechanism of colicin import through the bacterial outer membrane. Together with genetic analysis and studies on colicin occlusion of OmpF channels, this implied a colicin translocon consisting of BtuB and OmpF that would transfer the C-terminal cytotoxic domain (C96) of colicin E3 through the Escherichia coli outer membrane. This model does not, however, explain how the colicin attains the unfolded conformation necessary for transfer. Such a conformation change would require removal of the immunity (Imm) protein, which is bound tightly in a complex with the folded colicin E3. In the present study, it was possible to obtain reversible removal of Imm in vitro in a single column chromatography step without colicin denaturation. This resulted in a mostly unordered secondary structure of the cytotoxic domain and a large decrease in stability, which was also found in the receptor binding domain. These structure changes were documented by near- and far-UV circular dichroism and intrinsic tryptophan fluorescence. Reconstitution of Imm in a complex with C96 or colicin E3 restored the native structure. C96 depleted of Imm, in contrast to the native complex with Imm, efficiently occluded OmpF channels, implying that the presence of tightly bound Imm prevents its unfolding and utilization of the OmpF porin for subsequent import of the cytotoxic domain.

Primary events in the colicin translocon: FRET analysis of colicin unfolding initiated by binding to BtuB and OmpF.

Cellular import of colicin E3 is initiated by high affinity binding of the colicin receptor-binding (R) domain to the vitamin B(12) (BtuB) receptor in the Escherichia coli outer membrane. The BtuB binding site, at the apex of its extended coiled-coil R-domain, is distant from the C-terminal nuclease domain that must be imported for expression of cytotoxicity. Based on genetic analysis and previously determined crystal structures of the R-domain bound to BtuB, and of an N-terminal disordered segment of the translocation (T) domain inserted into the OmpF porin, a translocon model for colicin import has been inferred. Implicit in the model is the requirement for unfolding of the colicin segments inserted into OmpF. FRET analysis was employed to study colicin unfolding upon interaction with BtuB and OmpF. A novel method of Cys-specific dual labeling of a native polypeptide, which allows precise placement of donor and acceptor fluorescent dyes on the same polypeptide chain, was developed. A decrease in FRET efficiency between the translocation and cytotoxic domains of the colicin E3 was observed upon colicin binding in vitro to BtuB or OmpF. The two events were independent and additive. The colicin interactions with BtuB and OmpF have a major electrostatic component. The R-domain Arg399 is responsible for electrostatic interaction with BtuB. It is concluded that free energy for colicin unfolding is provided by binding of the R- domain to BtuB and binding/insertion of the T-domain to/into OmpF.

Minimum Length Requirement of the Flexible N-Terminal Translocation Subdomain of Colicin E3

The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.

Polylysine-Mediated Translocation of the Diphtheria Toxin Catalytic Domain through the Anthrax Protective Antigen Pore

The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores in the endosomal membrane, which translocate the effector proteins of the toxin to the cytosol. Effector proteins bind to oligomeric PA via their respective N-terminal domains and undergo N- to C-terminal translocation through the pore. Earlier we reported that a tract of basic amino acids fused to the N-terminus of an unrelated effector protein (the catalytic domain diphtheria toxin, DTA) potentiated that protein to undergo weak PA-dependent translocation. In this study, we varied the location of the tract (N-terminal or C-terminal) and the length of a poly-Lys tract fused to DTA and examined the effects of these variations on PA-dependent translocation into cells and across planar bilayers in vitro. Entry into cells was most efficient with ∼12 Lys residues (K12) fused to the N-terminus but also occurred, albeit 10–100-fold less efficiently, with a C-terminal tract of the same length. Similarly, K12 tracts at either terminus occluded PA pores in planar bilayers, and occlusion was more efficient with the N-terminal tag. We used biotin-labeled K12 constructs in conjunction with streptavidin to show that a biotinyl-K12 tag at either terminus is transiently exposed to the trans compartment of planar bilayers at 20 mV; this partial translocation in vitro was more efficient with an N-terminal tag than a C-terminal tag. Significantly, our studies with polycationic tracts fused to the N- and C-termini of DTA suggest that PA-mediated translocation can occur not only in the N to C direction but also in the C to N direction.