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1.C.10 The Pore-forming Haemolysin E (HlyE) Family

The HlyE family consists of a single protein, HlyE, and its close orthologues from species of Escherichia, Shigella and Salmonella. The E. coli protein is a functionally well characterized, pore-forming chromosomally-encoded haemolysin also called ClyA (cytolysin A), Hpr, and silent haemolysin, SheA. It consists of 303 amino acyl residues (34 kDa). Its transcription is positively controlled by SlyA, a regulator found in several enteric bacteria. HlyE forms stable, moderately cation-selective transmembrane pores with a diameter of 2.5-3.0 nm in lipid bilayers. The protein binds cholesterol, and pore formation in a membrane is stimulated if the membrane contains cholesterol. It forms oligomeric assemblies in the membrane (Wai et al., 2003).

The crystal structure of soluble E. coli HlyE has been solved to 2.0 Å resolution, and visualization of the lipid-associated form of the toxin at low resolution has been achieved by electron microscopy. The structure is different from other toxins, exhibiting an elaborate helical bundle some 100 Å long. It oligomerizes in the presence of lipid transmembrane to form predominantly octameric or dodecameric pores (Tzokov et al., 2006; Ludwig et al., 2010). The complexes are conformationally variable. The pores are longer than expected from the dimensions of the soluble protein suggesting that conformational changes occur on pore formation (Tzokov et al., 2006). HlyE protomers retain an α-helical structure when oligomerized to form a pore consisting of parallel HlyE protomers (Hunt et al., 2008).

The HlyE cytotoxin has recently been shown to be exported from the bacterium and assembled in vesicles derived from the outer membrane of E. coli (Wai et al., 2003). It forms oligomeric pore assemblies in the outer membrane, and the toxic activity of this outer membrane assembled toxin towards animal cells proved to be higher than that of the purified protein. Thus, outer bacterial membrane vesicles contribute to the activation and delivery of toxins (Wai et al., 2003).

The soluble monomer of ClyA must undergo large conformational changes to form the transmembrane pore. Mueller et al., 2009 reported the 3.3 A crystal structure of the 400 kDa dodecameric transmembrane pore formed by ClyA (HlyE). The tertiary structure of ClyA protomers in the pore is substantially different from that in the soluble monomer. The conversion involves more than half of all residues. It results in large rearrangements, up to 140 A, of parts of the monomer, reorganization of the hydrophobic core, and transitions of beta-sheets and loop regions to alpha-helices. The large extent of interdependent conformational changes indicates a sequential mechanism for membrane insertion and pore formation. 

ClyA, an α-pore forming toxin from pathogenic Escherichia coli (E. coli) and Salmonella enterica, assembles into an oligomeric structure in solution in the absence of either bilayer membranes or detergents at physiological temperature (Fahie et al. 2013). These oligomers can rearrange to create transmembrane pores when in contact with detergents or biological membranes. Intrinsic fluorescence measurements revealed that oligomers adopted an intermediate state found during the transition between monomer and transmembrane pore, suggesting that the water-soluble oligomer represents a prepore intermediate state.  Moreover, ClyA does not form transmembrane pores on E. coli lipid membranes. Because ClyA is delivered to the target host cell in an oligomeric conformation within outer membrane vesicles (OMVs), ClyA apparently forms a prepore oligomeric structure independently of the lipid membrane within the OMV, a non-classical pathway to attack eukaryotic host cells (Fahie et al. 2013).

The generalized transport reaction catalyzed by HlyE is:

small molecules (in) small molecules (out).

 

References associated with 1.C.10 family:

Del Castillo, F.J., S.C. Leal, F. Moreno, and I. Del Castillo. (1997). The Escherichia coli K-12 sheA gene encodes a 34-kDA secreted haemolysin. Mol. Microbiol. 25: 107-115. 11902713
Fahie, M., F.B. Romano, C. Chisholm, A.P. Heuck, M. Zbinden, and M. Chen. (2013). A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A. J. Biol. Chem. 288: 31042-31051. 24019520
Hunt, S., A.J. Moir, S. Tzokov, P.A. Bullough, P.J. Artymiuk, and J. Green. (2008). The formation and structure of Escherichia coli K-12 haemolysin E pores. Microbiol. 154: 633-642. 18227266
Ludwig, A., G. Völkerink, C. von Rhein, S. Bauer, E. Maier, B. Bergmann, W. Goebel, and R. Benz. (2010). Mutations affecting export and activity of cytolysin A from Escherichia coli. J. Bacteriol. 192: 4001-4011. 20511497
Ludwig, A., S. Bauer, R. Benz, B. Bergmann, and W. Goebel. (1999). Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12. Mol. Microbiol. 31: 557-567. 10027972
Mueller M., Grauschopf U., Maier T., Glockshuber R. and Ban N. (2009). The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism. Nature. 459(7247):726-30. 19421192
Oscarsson J., Y. Mizunoe, L. Li, X.H. Lai, A. Wieslander, and B.E. Uhlin. (1999). Molecular analysis of the cytolytic protein ClyA (SheA) from Echerichia coli. Mol. Microbiol. 32: 1226-1238. 10383763
Tzokov, S.B., N.R. Wyborn, T.J. Stillman, S. Jamieson, N. Czudnochowski, P.J. Artymiuk, J. Green, and P.A. Bullough. (2006). Structure of the hemolysin E (HlyE, ClyA, and SheA) channel in its membrane-bound form. J. Biol. Chem. 281: 23042-23049. 16754675
Wai, S.N., B. Lindmark, T. Soderblom, A. Takade, M. Westermark, J. Oscarsson, J. Jass, A. Richter-Dahlfors, Y. Mizunoe, and B.E. Uhlin. (2003). Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 115: 25-35. 14532000
Wai, S.N., M. Westermark, J. Oscarsson, J. Jass, E. Maier, R. Benz, and B.E. Uhlin. (2003). Characterization of dominantly negative mutant ClyA cytotoxin proteins in Escherichia coli. J. Bacteriol. 185: 5491-5499. 12949101
Wallace, A.J., T.J. Stillman, A. Atkins, S.J. Jamieson, P.A. Bullough, J. Green, and P.J. Artymiuk. (2000). E. coli hemolysin E (HlyE, ClyA, SheA): x-ray crystal structure of the toxin and observation of membrane pores by electron microscopy. Cell 100: 265-276. 10660049
Yadav, S.P., A. Ahmad, B.K. Pandey, D. Singh, N. Asthana, R. Verma, R.K. Tripathi, and J.K. Ghosh. (2009). A peptide derived from the putative transmembrane domain in the tail region of E. coli toxin hemolysin E assembles in phospholipid membrane and exhibits lytic activity to human red blood cells: plausible implications in the toxic activity of the protein. Biochim. Biophys. Acta. 1788: 538-550. 19111524