Structure-function of cyanobacterial outer-membrane protein, Slr1270: homolog of Escherichia coli drug export/colicin import protein, TolC.
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ABSTRACT:
SUBMITTER: Agarwal R
PROVIDER: S-EPMC4288923 | biostudies-literature | 2014 Nov
REPOSITORIES: biostudies-literature
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Project description:The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.
| S-EPMC1304901 | biostudies-other
Project description:The mechanism by which the drug export protein TolC is utilized for import of the cytotoxin colicin E1 across the outer membrane and periplasmic space is addressed. Studies of the initial binding of colicin E1 with TolC, occlusion of membrane-incorporated TolC ion channels, and the structure underlying the colicin-TolC complex were based on the interactions with TolC of individual colicin translocation domain (T-domain) peptides from a set of 19 that span different segments of the T-domain. These studies led to identification of a short 20-residue segment 101-120, a "TolC box", located near the center of the colicin T-domain, which is necessary for binding of colicin to TolC. Omission of this segment eliminated the ability of the T-domain to occlude TolC channels and to co-elute with TolC on a size-exclusion column. Far-ultraviolet circular dichroism spectral and thermal stability analysis of the structure of T-domain peptides implies (i) a helical hairpin conformation of the T-domain, (ii) the overlap of the TolC-binding site with a hinge of the helical hairpin, and (iii) a TolC-dependent stage of colicin import in which a central segment of the T-domain in a helical hairpin conformation binds to the TolC entry port following initial binding to the BtuB receptor. These studies provide the first structure-based information about the interaction of colicin E1 with the unique TolC protein. The model inferred for binding of the T-domain to TolC implies reservations about the traditional model for colicin import in which TolC functions to provide a channel for translocation of the colicin in an unfolded state across the bacterial outer membrane and a large part of the periplasmic space.
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Project description:TolC is a member of the outer membrane efflux proteins (OEPs) family and acts as an exit duct to export proteins, antibiotics, and substrate molecules across the Escherichia coli cell membrane. Export of these molecules is evidenced to be brought about through the reversible interactions and binding of substrate-specific drug molecules or antibiotics with TolC and by being open for transport, which afterward leads to cross-resistance. Hence, the binding of kanamycin with TolC was monitored through molecular docking (MD), the structural fluctuations and conformational changes to the atomic level. The results were further supported from the steady-state fluorescence binding and isothermal titration calorimetry (ITC) studies. Binding of kanamycin with TolC resulted in a concentration dependent fluorescence intensity quenching with 7 nm blue shift. ITC binding data maintains a single binding site endothermic energetic curve with binding parameters indicating an entropy driven binding process. The confirmational changes resulting from this binding were monitored by a circular dichroism (CD) study, and the results showed insignificant changes in the α-helix and β-sheets secondary structure contents, but the tertiary structure shows inclusive changes in the presence of kanamycin. The experimental data substaintially correlates the RMSD, R g, and RMSF results. The resulting conformational changes of the TolC-kanamycin complexation was stabilized through H-bonding and other interactions.
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Project description:The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps, the most fascinating transport phenomenon is the import and export of very large protein toxins using relatively small β-barrel proteins residing in the outer membrane. Progress has been made on three systems in recent years that shed light on this process. In this review, we summarize bacteriocin (toxin) import using TonB-dependent transporters and protein secretion by autotransporters and two partner secretion systems.
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Project description:The outer membrane protein (OMP) TolC is the cell surface component of several drug efflux pumps that are responsible for bacterial resistance against a variety of antibiotics. In this research, we investigated the effects of OMP TolC on E. coli transport within saturated sands through column experiments using a wild-type E. coli K12 strain (with OMP TolC), as well as the corresponding transposon mutant (tolC::kan) and the markerless deletion mutant (?tolC). Our results showed OMP TolC could significantly enhance the transport of E. coli when the ionic strength was 20 mM NaCl or higher. The deposition rate coefficients for the wild-type E. coli strain (with OMP TolC) was usually >50% lower than those of the tolC-negative mutants. The measurements of contact angles using three probe liquids suggested that TolC altered the surface tension components of E. coli cells and lead to lower Hamaker constants for the cell-water-sand system. The interaction energy calculations using the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory suggested that the deposition of the E. coli cell primarily occurred at the secondary energy minimum. The depth of the secondary energy minimum increased with ionic strength, and was greater for the TolC-deletion strains under high ionic strength conditions. Overall, the transport behavior of three E. coli strains within saturated sands could be explained by the XDLVO calculations. Results from this research suggested that antibiotic resistant bacteria expressing OMP TolC could spread more widely within sandy aquifers.
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Project description:Analysis of the nucleotide sequence of an Escherichia coli colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.
| S-EPMC93827 | biostudies-literature
Project description:Escherichia coli excretes the catecholate siderophore enterobactin in response to iron deprivation. While the mechanisms underlying enterobactin biosynthesis and ferric enterobactin uptake and utilization are widely understood, nearly nothing is known about how enterobactin is exported from the cell. Mutant and high-performance liquid chromatography analyses demonstrated that the outer membrane channel tunnel protein TolC but none of the respective seven resistance nodulation cell division (RND) proteins CusA, AcrB, AcrD, AcrF, MdtF (YhiV), or the twin RND MdtBC (YegNO) was essential for enterobactin export across the outer membrane. Mutant E. coli strains with additional deletion of tolC or the major facilitator entS were growth deficient in iron-depleted medium. Strains with deletion of tolC or entS, but not with deletion of genes encoding RND transporters, excreted very little enterobactin into the growth medium. Enterobactin excretion in E. coli is thus probably a two-step process involving the major facilitator EntS and the outer membrane channel tunnel protein TolC. Quantitative reverse transcription-PCR analysis of gene-specific transcripts showed no significant changes in tolC expression upon iron depletion. However, iron starvation led to increased expression of the RND gene mdtF and a decrease in acrD.
| S-EPMC1251586 | biostudies-literature
Project description:Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.
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Project description:Colicin M (Cma) is a protein toxin produced by Escherichia coli that kills sensitive E. coli cells by inhibiting murein biosynthesis in the periplasm. Recombinant plasmids carrying cbrA (formerly yidS) strongly increased resistance of cells to Cma, whereas deletion of cbrA increased Cma sensitivity. Transcription of cbrA is positively controlled by the two-component CreBC system. A ΔcreB mutant was highly Cma sensitive because little CbrA was synthesized. Treatment of CbrA-overproducing cells by osmotic shock failed to render cells Cma sensitive because the cells were resistant to osmotic shock. In a natural environment with a growth-limiting nutrient supply, cells producing CbrA defend themselves against colicin M synthesized by competing cells. Isolated CbrA is a protein with noncovalently bound flavin adenine dinucleotide. Sequence comparison and structure prediction assign the closest relative of CbrA with a known crystal structure as digeranylgeranyl-glycerophospholipid reductase of Thermoplasma acidophilum. CbrA is found in Escherichia coli, Citrobacter, and Salmonella bongori but not in other enterobacteria. The next homologs with the highest identity (over 50%) are found in the anaerobic Clostridium botulinum group 1 and a few other Firmicutes.
| S-EPMC3430330 | biostudies-literature
Project description:Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A colicins.
| S-EPMC7335789 | biostudies-literature