title: Interdomain Ca(2+) effects in Escherichia coli alpha-haemolysin: Ca(2+) binding to the C-terminal domain stabilizes both C- and N-terminal domains.
abstract: alpha-Haemolysin (HlyA) is a toxin secreted by pathogenic Escherichia coli, whose lytic activity requires submillimolar Ca(2+) concentrations. Previous studies have shown that Ca(2+) binds within the Asp and Gly rich C-terminal nonapeptide repeat domain (NRD) in HlyA. The presence of the NRD puts HlyA in the RTX (Repeats in Toxin) family of proteins. We tested the stability of the whole protein, the amphipathic helix domain and the NRD, in both the presence and absence of Ca(2+) using native HlyA, a truncated form of HlyADeltaN601 representing the C-terminal domain, and a novel mutant HlyA W914A whose intrinsic fluorescence indicates changes in the N-terminal domain. Fluorescence and infrared spectroscopy, tryptic digestion, and urea denaturation techniques concur in showing that calcium binding to the repeat domain of alpha-haemolysin stabilizes and compacts both the NRD and the N-terminal domains of HlyA. The stabilization of the N-terminus through Ca(2+) binding to the C-terminus reveals long-range inter-domain structural effects. Considering that RTX proteins consist, in general, of a Ca(2+)-binding NRD and separate function-specific domains, the long-range stabilizing effects of Ca(2+) in HlyA may well be common to other members of this family.
authors.affiliation: Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and the Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Lissete
authors.initials: L
authors.lastname: Sánchez-Magraner
authors.forename: Aitziber L
authors.initials: AL
authors.lastname: Cortajarena
authors.forename: Marcos
authors.initials: M
authors.lastname: García-Pacios
authors.forename: José-Luis R
authors.initials: JL
authors.lastname: Arrondo
authors.forename: Jon
authors.initials: J
authors.lastname: Agirre
authors.forename: Diego M A
authors.initials: DM
authors.lastname: Guérin
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: Helena
authors.initials: H
authors.lastname: Ostolaza
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D002118:Calcium