title: His-859 is an essential residue for the activity and pH dependence of Escherichia coli RTX toxin alpha-hemolysin.
abstract: Escherichia coli alpha-hemolysin (HlyA) is a toxin protein that, in common with other members of the RTX family, contains a calcium-binding domain consisting of a number of Gly- and Asp-rich nonapeptides (17 in this case) repeated in tandem. Amino acid number 6 in these nonapeptides is almost invariably Asp, and occasionally Asn, but HlyA contains a His residue (number 859 in the chain) in position 6 of the last-but-one nonapeptide. HlyA mutants have been prepared, by site-directed mutagenesis, in which His-859 has been replaced by an Asn (H859N) or by Asp (H859D). HlyA exists in aqueous media in an aggregate-monomer equilibrium, but only the monomer containing bound Ca(2+) (HlyA.Ca) appears to be competent to achieve target membrane insertion and subsequent lysis. In mutant H859N, equilibrium appears to be shifted toward the aggregate, therefore the protein does not exchange Ca(2+) with the aqueous environment, no HlyA.Ca monomers are detected, and the protein lacks any membrane lytic activity. Mutant H859D in turn is almost indistinguishable from the wild-type regarding its calcium binding and membrane lytic activity, however, it differs significantly in its pH dependence. Wild-type HlyA activity decreases sigmoidally with pH, following rather closely the protonation curve of a His residue (apparent pK(a) approximately 6.5). With mutant H859D activity decreases almost linearly with pH and to a smaller extent. It can be concluded that His-859 plays a critical role in several aspects of HlyA activity, namely self-aggregation properties, calcium binding, hemolysis, and pH dependence.
authors.affiliation: Unidad de Biofísica (CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Aitziber L
authors.initials: AL
authors.lastname: Cortajarena
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: Helena
authors.initials: H
authors.lastname: Ostolaza
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000502:physiology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers; D006639:Histidine; D002118:Calcium