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-A coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 A is reported. Binding of R135 to the BtuB extracellular surface (DeltaG(o) = -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.     

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.     

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.     

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-A 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. Arg(452) 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.     

Structural stability and domain organization of colicin E1

Thermodynamic properties, stability, and structure of the toxin-like molecule colicin E1 were analyzed by differential scanning calorimetry and circular dichroism to determine the number of structurally independent domains, and the interdomain interactions necessary for colicin import into the Escherichia coli cell. Analysis of denaturation profiles of the 522 residue colicin E1, together with fragments of 342 and 178 residues that contain subsets of the domains, showed three stable cooperative blocks that differ in thermal stability and correspond to three major functional domains of the colicin: (i) the COOH-terminal channel-forming (C) domain with the highest thermal stability; (ii) the BtuB receptor binding (R) domain; and (iii) the N-terminal translocation (T) domain that has the smallest stabilization enthalpy and thermal stability. Interdomain interactions were described in which T-R interactions stabilize R, and T-C and R-C interactions stabilize R and T, but destabilize C. The R and T domains behaved in a similar way as a function of pH and ionic strength. Interacting extended helices of the R domain, possibly a coiled-coil, were implied by: (i) the very high (>90%) alpha-helical content of the R domain, (ii) cooperative decreases in alpha-helical content near the T(tr) of thermal denaturation of the R domain; (iii) a large denaturation enthalpy, implying extensive H-bond and van der Waals interactions. The R domain was inferred, from the extended network of interacting helices, large DeltaH, and steep temperature dependence of its stabilization energy to have a dominant role in determining the conformation of other domains. It is proposed that cellular import starts with the R domain binding to the BtuB receptor, followed by unfolding of the R domain coiled-coil and thereby of the T domain, which then interacts with the TolC receptor-translocator.     

Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9(s-s)) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9(s-s), we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9(AF)) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.     

Multiple extracellular loops contribute to substrate binding and transport by the Escherichia coli cobalamin transporter BtuB

The Escherichia coli outer membrane TonB-dependent transporters for iron complexes and cobalamins recognize their multiple and diverse substrates with high specificity and affinity. The X-ray crystallographic structures of several transporters show that the substrate-binding surfaces are comprised of residues from the internal globular domain and multiple extracellular loops. The extracellular loops on the N-terminal half of the transmembrane beta-barrel of the cobalamin transporter BtuB participate in binding of the cofactor calcium atoms and undergo substantial conformation changes upon substrate binding. The functional relevance of the five C-terminal loops was examined by examining the effects of short in-frame deletions. Each loop contributed in different ways to the binding of BtuB substrates. Deletions in loops 7, 8, 9, and 11 strongly decreased cobalamin binding and transport, whereas deletions in loops 8, 9, and 10 affected binding and entry of phage BF23. None of the loops were essential for the action of colicin E1 or E3, which is consistent with the crystallographic observation that the colicin E3 receptor-binding domain can contact almost all of the loops. A deletion in loop 9 or 11 eliminated the ability of cobalamin to inhibit the action of colicin E1. These phenotypes show that there are multiple independent binding elements and point out similarities and differences in binding properties among the TonB-dependent transporters.     

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 A 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 A 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.     

Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.     

Metal-induced folding of a designed metalloprotein

The metal-induced assembly of a designed peptide-based rubredoxin model is described. The C16C19-GGY peptide has the sequence Ac-K(IEALEGK)(2)(CEACEGK)(IEALEGK)GGY-amide in which the presence of the Cys-X-X-Cys metal binding domain of rubredoxin was used to place cysteine residues at the hydrophobic "a" and "d" positions upon formation of a homodimeric alpha-helical coiled-coil. Circular dichroism spectroscopy shows that the apopeptide exists as a random coil and assembles into a coiled-coil in the presence of Cd(2+). Metal binding is monitored by the appearance of a new LMCT band at 238 nm. UV-Vis titrations and SDS-PAGE experiments are used to show that this designed metalloprotein exists as a metal-bridged coiled-coil dimer.     

Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coli

Colicins A, E1, E2 and E3 belong to the BtuB group of colicins. The NH2-terminal region of colicin A is required for translocation, and defects in this region cannot be overcome by osmotic shock of sensitive cells. In addition to BtuB, colicin A requires OmpF for efficient uptake by sensitive cells. The roles of BtuB and OmpF in translocation and binding to the receptor of the colicins A, E1, E2 and E3 were compared. The results suggest that for colicin A OmpF is used both as a receptor and for translocation across the outer membrane. In contrast, for colicin E1, OmpF is used neither as a receptor nor for translocation. For colicins E2 and E3, the situation is intermediate: only BtuB is used as a receptor but both BtuB and OmpF are involved in the translocation step.     

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.     

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.     

Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution

Colicin B (55 kDa) is a cytotoxic protein that recognizes the outer membrane transporter, FepA, as a receptor and, after gaining access to the cytoplasmic membranes of sensitive Escherichia coli cells, forms a pore that depletes the electrochemical potential of the membrane and ultimately results in cell death. To begin to understand the series of dynamic conformational changes that must occur as colicin B translocates from outer membrane to cytoplasmic membrane, we report here the crystal structure of colicin B at 2.5 A resolution. The crystal belongs to the space group C2221 with unit cell dimensions a = 132.162 A, b = 138.167 A, c = 106.16 A. The overall structure of colicin B is dumbbell shaped. Unlike colicin Ia, the only other TonB-dependent colicin crystallized to date, colicin B does not have clearly structurally delineated receptor-binding and translocation domains. Instead, the unique N-terminal lobe of the dumbbell contains both domains and consists of a large (290 residues), mostly beta-stranded structure with two short alpha-helices. This is followed by a single long ( approximately 74 A) helix that connects the N-terminal domain to the C-terminal pore-forming domain, which is composed of 10 alpha-helices arranged in a bundle-type structure, similar to the pore-forming domains of other colicins. The TonB box sequence at the N-terminus folds back to interact with the N-terminal lobe of the dumbbell and leaves the flanking sequences highly disordered. Comparison of sequences among many colicins has allowed the identification of a putative receptor-binding domain.     

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.     

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.     

Binding domains of colicins E1, E2 and E3 in the receptor protein btub ofEscherichia coli

Eight reagents specifically modifying amino acids were applied to cells of a standardEscherichia coli colicin indicator strain to followin vivo changes of its binding capacity for colicins E1–E3 and hence the binding domains (epitopes) for them in the outer membrane receptor protein BtuB. The effect of these reagents was also investigated in a mutant strain carrying an extensive BtuB deletion. The following differences of the binding epitopes could be ascertained.Colicin E1: Blockage of OH-groups, just as N-substitution of His and modification of Arg and Trp enhance binding of colicin E1. In the deleted receptor, also abolition of carboxylic anion bonds enhances its affinity for colicin E1. It follows that colicin E1 is bound, most of all, to the hydrophobic domain A (loops 1+2) of BtuB.Colicins E2 and E3: both exert rather analogous binding parameters. In contrast to E1, O-substitution of Ser and Thr dramatically decreases the E2 and E3 binding, similarly to modification of Lys. There is also a clear difference in the binding affinity of the domain for E2 and/or E3 and for E1 following modifications of their Arg and His. Colicins E2 and E3 are rather bound to the hydrophilic domain B (loops 5–7) of the receptor. In this respect, interactions of colicins E2 and E3 with deeper parts of A and B domains (Trp, several Arg, Lys and His residues) exhibited subtle differences. Acidic pH (4.5–6.0) shows a positive, while pH 7.0–8.5 a rather negative impact on the receptor-binding function for the colicins. It was clearly demonstrated that there is just a partial difference between the binding behavior of colicins E1, E2 and/or E3.     

Epitope mapping and direct visualization of the parallel, in-register arrangement of the double-stranded coiled-coil in the NuMA protein.

NuMA, a 238 kDa protein present in the nucleus during interphase, translocates to the spindle poles in mitosis. NuMA plays an essential role in mitosis, since microinjection of the NuMA SPN-3 monoclonal antibody causes mitotic arrest and micronuclei formation. We have mapped the approximate position of the epitopes of six monoclonal NuMA antibodies using recombinant NuMA fragments. The SPN-3 epitope has been located to residues 255-267 at the C-terminus of the first helical subdomain of the central rod domain and several residues crucial for antibody binding have been identified. To gain insight into the ultrastructure of NuMA, several defined fragments, as well as the full-length recombinant protein, were expressed in Escherichia coli and purified to homogeneity. They were then characterized by chemical cross-linking, circular dichroism spectra and electron microscopy. The results directly reveal the tripartate structure of NuMA. A long central rod domain is flanked by globular end domains. The rod is 207 nm long and is at least 90% alpha-helical. It reflects a double-stranded coiled-coil with the alpha-helices arranged parallel and in register. The NuMA protein thus forms the longest coiled-coil currently known. Our analyses reveal no indication that recombinant NuMA assembles into filaments or other higher order structures.     

Presentation of epitopes on genetically engineered peptides and selection of lymphoma-targeting moieties based on epitope biorecognition

A His-tagged coiled coil stem loop peptide with stable secondary structure was designed and biosynthesized. A series of oligopeptides related to the EBV envelope glycoprotein 350/220 N-terminal nonapeptide as potential CD21 receptor-binding epitopes were engineered into the loop region of the peptide scaffold. It was shown that these peptides had a stable alpha-helical coiled coil structure and assumed a monomeric form in PBS. Biorecognition of the epitopes was studied by immobilizing the epitope-containing peptides on complexed Ni2+-containing surfaces through His-Ni2+ chelation and incubating with purified soluble CD21 receptor or CD21+ cells. The results showed that the potential epitopes bound to CD21 and CD21+ cells at different affinities depending on oligopeptide structures. This approach allows for the evaluation of epitope biorecognizabilities and the selection of optimal oligopeptides among sequences for use as targeting moieties in the design of new lymphoma-targeting polymeric drug carriers.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.