title: The calcium-binding C-terminal domain of Escherichia coli alpha-hemolysin is a major determinant in the surface-active properties of the protein.
abstract: alpha-Hemolysin (HlyA) from Escherichia coli is a protein toxin (1024 amino acids) that targets eukaryotic cell membranes, causing loss of the permeability barrier. HlyA consists of two main regions, an N-terminal domain rich in amphipathic helices, and a C-terminal Ca(2+)-binding domain containing a Gly- and Asp-rich nonapeptide repeated in tandem 11-17 times. The latter is called the RTX domain and gives its name to the RTX protein family. It had been commonly assumed that membrane interaction occurred mainly if not exclusively through the amphipathic helix domain. However, we have cloned and expressed the C-terminal region of HlyA, containing the RTX domain plus a few stabilizing sequences, and found that it is a potent surface-active molecule. The isolated domain binds Ca(2+) with about the same affinity (apparent K(0.5) approximately 150 microM) as the parent protein HlyA, and Ca(2+) binding induces in turn a more compact folding with an increased proportion of beta-sheet structure. Both with and without Ca(2+) the C-terminal region of HlyA can interact with lipid monolayers spread at an air-water interface. However, the C-terminal domain by itself is devoid of membrane lytic properties. The present results can be interpreted in the light of our previous studies that involved in receptor binding a peptide in the C-terminal region of HlyA. We had also shown experimentally the distinction between reversible membrane adsorption and irreversible lytic insertion of the toxin. In this context, the present data allow us to propose that both major domains of HlyA are directly involved in membrane-toxin interaction, the nonapeptide repeat, calcium-binding RTX domain being responsible for the early stages of HlyA docking to the target membrane.
authors.affiliation: Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Lissete
authors.initials: L
authors.lastname: Sánchez-Magraner
authors.forename: Ana R
authors.initials: AR
authors.lastname: Viguera
authors.forename: Marcos
authors.initials: M
authors.lastname: García-Pacios
authors.forename: M Pilar
authors.initials: MP
authors.lastname: Garcillán
authors.forename: José-Luis R
authors.initials: JL
authors.lastname: Arrondo
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: Helena
authors.initials: H
authors.lastname: Ostolaza
mesh_terms: D000327:Adsorption; D000388:Air; D002118:Calcium / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D014867:Water / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008055:Lipids; D008081:Liposomes; D014867:Water; D002118:Calcium