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Structure and function of colicin S4, a colicin with a duplicated receptor-binding domain.

ABSTRACT: Colicins are plasmid-encoded toxic proteins produced by Escherichia coli strains to kill other E. coli strains that lack the corresponding immunity protein. Colicins intrude into the host cell by exploiting existing transport, diffusion, or efflux systems. We have traced the way colicin S4 takes to execute its function and show that it interacts specifically with OmpW, OmpF, and the Tol system before it inserts its pore-forming domain into the cytoplasmic membrane. The common structural architecture of colicins comprises a translocation, a receptor-binding, and an activity domain. We have solved the crystal structure of colicin S4 to a resolution of 2.5 A, which shows a remarkably compact domain arrangement of four independent domains, including a unique domain duplication of the receptor-binding domain. Finally, we have determined the residues responsible for binding to the receptor OmpW by mutating exposed charged residues in one or both receptor-binding domains.

SUBMITTER: Arnold T

PROVIDER: S-EPMC2649078 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Structure and function of colicin S4, a colicin with a duplicated receptor-binding domain.

Arnold Thomas T Zeth Kornelius K Linke Dirk D

The Journal of biological chemistry 20081204 10

Colicins are plasmid-encoded toxic proteins produced by Escherichia coli strains to kill other E. coli strains that lack the corresponding immunity protein. Colicins intrude into the host cell by exploiting existing transport, diffusion, or efflux systems. We have traced the way colicin S4 takes to execute its function and show that it interacts specifically with OmpW, OmpF, and the Tol system before it inserts its pore-forming domain into the cytoplasmic membrane. The common structural architec ...[more]

PMID: 19056731

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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.

Project description:The emergence of multidrug-resistant Escherichia coli, which poses a major threat to public health, has motivated the development of numerous alternative antimicrobials. Lysins are bacteriophage- and bacterium-derived peptidoglycan hydrolases that represent a new antibiotic treatment targeting bacterial cell walls. However, the bactericidal effect of native lysins on Gram-negative bacteria is restricted by the presence of an outer membrane. Here, we first evaluated the antibacterial activity of three Campylobacter-derived lysins (Clysins) against E. coli. To improve their transmembrane ability and antibacterial activities, six engineered Clysins were constructed by fusing with the translocation and receptor-binding (TRB) domains from two types of colicins (colicin A [TRBA] and colicin K [TRBK]), and their biological activities were determined. Notably, engineered lysin TRBK-Cly02 exhibited the highest bactericidal activity against the E. coli BL21 strain, with a reduction of 6.22 ± 0.34 log units of cells at a concentration of 60.1 μg/mL, and formed an observable inhibition zone even at a dose of 6.01 μg. Moreover, TRBK-Cly02 killed E. coli dose dependently and exhibited the strongest bactericidal activity at pH 6. It also exhibited potential bioactivity against multidrug-resistant E. coli clinical isolates. In summary, this study identified three lysins from Campylobacter strains against E. coli, and the enhancement of their antibacterial activities by TRB domains fusion may allow them to be developed as potential alternatives to antibiotics. IMPORTANCE Three lysins from Campylobacter, namely, Clysins, were investigated, and their antibacterial activities against E. coli were determined for the first time. To overcome the restriction of the outer membrane of Gram-negative bacteria, we combined the TRB domains of colicins with these Clysins. Moreover, we discovered that the Clysins fused with TRB domains from colicin K (TRBK) killed E. coli more effectively, and this provides a new foundation for the development of novel bioengineered lysins by employing TRBK constructs that target outer membrane receptor/transport systems. One of the designed lysins, TRBK-Cly02, exhibited potent bactericidal efficacy against E. coli strains and may be used for control of multidrug-resistant clinical isolates. The results suggest that TRBK-Cly02 can be considered a potential antibacterial agent against pathogenic E. coli.

Dataset Information

Structure and function of colicin S4, a colicin with a duplicated receptor-binding domain.

Publications

Structure and function of colicin S4, a colicin with a duplicated receptor-binding domain.

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