Interaction of Escherichia coli hemolysin with biological membranes

Abstract

Escherichia coli hemolysin (HlyA) is a membrane-permeabilizing protein belonging to the family of RTX-toxins. Lytic activity depends on binding of Ca 21 to the C-terminus of the molecule. The N-terminus of HlyA harbors hydrophobic sequences that are believed to constitute the membrane-inserting domain. In this study, 13 HlyA cysteine-replacement mutants were constructed and labeled with the polarity-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The fluorescence emission of the label was examined in soluble and membrane-bound toxin. Binding effected a major blue shift in the emission of six residues within the N-terminal hydrophobic domain, indicating insertion of this domain into the lipid bilayer. The emission shifts occurred both in the presence and absence of Ca 21 , suggesting that Ca 21 is not required for the toxin to enter membranes. However, binding of Ca 21 to HlyA in solution effected conformational changes in both the C-terminal and N-terminal domain that paralleled activation. Our data indicate that binding of Ca 21 to the toxin in solution effects a conformational change that is relayed to the N-terminal domain, rendering it capable of adopting the structure of a functional pore upon membrane binding.Keywords: badan; calcium; conformation; Escherichia coli hemolysin, membrane.Escherichia coli hemolysin (HlyA), a 107-kDa protein of 1024 amino acids devoid of cysteine [1], belongs to the family of RTX-toxins [2] that are produced by many Gramnegative organisms. To obtain pore-forming activity, HlyA requires post-translational acylation of two lysine residues, Lys564 and Lys690 [3,4]. The protein harbors a hydrophobic domain between residues 177 and 411, which according to predictions of secondary structure [5] largely assumes helical conformation and might be involved in membrane penetration. The C-terminal half of the molecule contains 12 repeats of the consensus nonapeptide X-Leu-XGly-Gly-X-X-Gly-Asp-Asp-Asp. This repeat sequence spanning residues 739±849 represents a Ca 21 -binding domain and is essential for function [6±9]. In the presence of Ca 21 , HlyA forms pores of < 2 nm diameter in target membranes [10]. Several approaches to study structurefunction analysis of HlyA have been undertaken in the past. CD spectroscopy was used to investigate the effect of Ca 21 on HlyA, but no significant changes in secondary structure could be detected with this method [11]. On the other hand, results obtained from intrinsic fluorescence measurements and trypsin digestion were compatible with a Ca 21 -induced change in conformation [11]. Moayeri and Welch [12] studied the altered accessibility of toxin domains for monoclonal antibodies after the binding of HlyA to membranes. They reported that the far N-terminus and the region between residues 594 and 640 remain accessible in the membrane-bound toxin. No conclusions could be made for the putative transmembrane region and the C-terminal domain, as antibodies for these regions were not available.An important step toward unders...

Bacteria differentially induce degradation of Bcl-xL, a survival protein, by human platelets

“…28,31 Incubation of platelets with WAM582, a nonpathogenic K-12 laboratory strain of E coli that encodes for the wild-type hlyA operon, induced Bcl-x L degradation within 2 hours ( Figure 3C). In contrast, WAM783, which carries the hlyA operon lacking hlyC, failed to induce Bcl-x L degradation in incubations lasting up to 8 hours ( Figure 3C).…”

A Receptor-binding Region in Escherichia coli α-Haemolysin

“…Recent data indicate that insertion may take place in the absence of Ca 2ϩ (9,10), but Ca 2ϩ binding to the nonapeptide repeat domain is essential for membrane lysis (9,11,12). Note that the Ca 2ϩ -binding domain is located near the protein C terminus, whereas the membrane insertion domain is located near the N terminus (9,10,13). The present study is devoted to exploring the early stages of HlyA interaction with the target cell, namely its binding to the surface receptor.…”

“…HlyA first binds a receptor on the cell surface, a ␤ 2 -integrin in leukocytes (7) or glycophorin in red blood cells (8), and then becomes inserted in the cell membrane. Recent data indicate that insertion may take place in the absence of Ca 2ϩ (9,10), but Ca 2ϩ binding to the nonapeptide repeat domain is essential for membrane lysis (9,11,12). Note that the Ca 2ϩ -binding domain is located near the protein C terminus, whereas the membrane insertion domain is located near the N terminus (9,10,13).…”

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas’ disease

“…They are considered a virulence factor from these microorganisms and also a member of the RTX (repeats in toxin) protein family (Welch, 2001). The presence of amphipathic and hydrophilic domains confers to the protein an overall amphiphilic character, which explains its tendency both to aggregate and to interact with membranes (Hyland et al, 2001;Schindel et al, 2001;Soloaga et al, 1999).…”