Stanislav D. Zakharov

The structure of BtuB with bound colicin E3 R-domain implies a translocon

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-Å coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 Å is reported. Binding of R135 to the BtuB extracellular surface (ΔG° = −12 kcal mol−1) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135–BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.

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.

Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore

The channel activity of colicin E1 was studied in planar lipid bilayers and liposomes. Colicin E1 pore‐forming activity was found to depend on the curvature of the lipid bilayer, as judged by the effect on channel activity of curvature‐modulating agents. In particular, the colicin‐induced trans‐membrane current was augmented by lysophosphatidylcholine and reduced by oleic acid, agents promoting positive and negative membrane curvature, respectively. The data obtained imply direct involvement of lipids in the formation of colicin E1‐induced pore walls. It is inferred that the toroidal pore model previously validated for small antimicrobial peptides is applicable to colicin E1, a large protein that contains ten α‐helices in its pore‐forming domain.

Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes.

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.

Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed “small” conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel “large” conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 Å, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid “flip-flop” and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.

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.

ON THE MECHANISM AND PATHWAY OF COLICIN IMPORT ACROSS THE E. COLI OUTER MEMBRANE

Colicins and phages parasitize outer membrane receptors whose physiological purpose is the transport of metabolites, metals, vitamins, and sugars. From mutagenesis studies, it is known that several colicins require the function of two outer membrane protein (Omp) receptors for cytotoxicity. A formidable list of problems associated with an understanding of a two receptor mechanism for colicin translocation includes the definition of the sites of initial binding and interactions of the colicin with the OM translocator protein, the working lumenal aperture of the translocator, the question of whether the colicin must be unfolded for translocation, the source of energy for unfolding and translocation, the order of colicin translocation, and the sites and mechanism of interaction of the colicins with the Tol-Pal proteins on the periplasmic side of the outer membrane. 3D crystal structures recently obtained of the cobalamin (vitamin B12) receptor (BtuB), and of the complex of BtuB with the 135 residue receptor binding domain (R135) of colicin E3, have provided some new insights on the interactions between two Omp receptors that are necessary for translocation of colicins. Together with spectroscopic data on the R135-BtuB interaction and electrophysiological data on the colicin E3-OmpF interaction, this has led to a proposal for the utilization of two receptors, BtuB-OmpF, in an outer membrane translocon for colicin E3.

Membrane binding of the colicin E1 channel: activity requires an electrostatic interaction of intermediate magnitude.

In vitro channel activity of the C-terminal colicin E1 channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, I = 0.12 M, pH 4.0), the free energy of binding was delta G approximately -9 kcal/mol, with nonelectrostatic and electrostatic components, delta Gnel approximately -5 kcal/mol and delta Gel approximately -4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.

Structural insight into the role of the Ton complex in energy transduction

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron–electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

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.