Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal gamma-haemolysins in lipid membranes.
Staphylococcal gamma-haemolysins are bicomponent toxins in a family including other leucocidins and alpha-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 alpha-toxin, but different selectivity (cation instead of anion). Structural analogies between gamma-haemolysins and alpha-toxin indicate the presence, at the pore entry, of a conserved region containing four positive charges in alpha-toxin, but either positive or negative in gamma-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 alpha-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 gamma-haemolysin pores depend substantially on the presence and location of charged residues in the channel.
Comai
Massimiliano
M
CNR-ITC Centro di Fisica degli Stati Aggregati, Istituto di Biofisica del CNR, Via Sommarive 18, I-38050 Povo (Trento), Italy.
Dalla Serra
Mauro
M
Coraiola
Manuela
M
Werner
Sandra
S
Colin
Didier A
DA
Monteil
Henri
H
Prévost
Gilles
G
Menestrina
Gianfranco
G
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000187:drug effects; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D016415:Sequence Alignment; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C037956:gamma-hemolysin, Staphylococcus aureus