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Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal γ ‐haemolysins in lipid membranes
Author(s) -
Comai Massimiliano,
Serra Mauro Dalla,
Coraiola Manuela,
Werner Sandra,
Colin Didier A.,
Monteil Henri,
Prévost Gilles,
Menestrina Gianfranco
Publication year - 2002
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2002.02943.x
Subject(s) - membrane , conductance , selectivity , hemolysin , toxin , biophysics , mutant , divalent , biology , chemistry , biochemistry , organic chemistry , gene , mathematics , combinatorics , virulence , catalysis
Summary Staphylococcal γ‐haemolysins are bicomponent toxins in a family including other leucocidins and α‐toxin. Two active toxins are formed combining HlgA or HlgC with HlgB. Both open pores in lipid membranes with conductance, current voltage characteristics and stability similar to α‐toxin, but different selectivity (cation instead of anion). Structural analogies between γ‐haemolysins and α‐toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in α‐toxin, but either positive or negative in γ‐haemolysins. Four mutants were produced (HlgA D44K, HlgB D47K, HlgB D49K and HlgB D47K/D49K) converting those negative charges to positive in HlgA and HlgB. When all charges were positive, the pores had the same selectivity and conductance as α‐toxin, suggesting that the cluster may form an entrance electrostatic filter. As mutated HlgC‐HlgB pores were less affected, additional charges in the lumen of the pore were changed (HlgB E107Q, HlgB D121N, HlgB T136D and HlgA K108T). Removing a negative charge from the lumen made the selectivity of both HlgA‐HlgB D121N and HlgC‐HlgB D121N more anionic. Residue D121 of HlgB is compensated by a positive residue (HlgA K108) in the HlgA‐HlgB pore, but isolated in the more cation‐selective HlgC‐HlgB pore. Interestingly, the pore formed by HlgA K108T‐HlgB, in which the positive charge of HlgA was removed, was as cation selective as HlgC‐HlgB. Meanwhile, the pore formed by HlgA K108T‐HlgB D121N, in which the two charge changes compensated, retrieved the properties of wild‐type HlgA‐HlgB. We conclude that the conductance and selectivity of the γ‐haemolysin pores depend substantially on the presence and location of charged residues in the channel.