Protein engineering modulates the transport properties and ion selectivity of the pores formed by staphylococcal gamma-haemolysins in lipid membranes
| Autor: | Massimiliano, Comai, Mauro, Dalla Serra, Manuela, Coraiola, Sandra, Werner, Didier A, Colin, Henri, Monteil, Gilles, Prévost, Gianfranco, Menestrina |
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| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Erythrocytes Neutrophils Bacterial Toxins Lipid Bilayers Molecular Sequence Data Protein Engineering Hemolysis Ion Channels Protein Structure Secondary Protein Structure Tertiary Hemolysin Proteins Bacterial Proteins Spectroscopy Fourier Transform Infrared Mutagenesis Site-Directed Animals Humans Amino Acid Sequence Rabbits Sequence Alignment |
| Zdroj: | Molecular microbiology. 44(5) |
| ISSN: | 0950-382X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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