title: Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59.
abstract: The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.
authors.affiliation: Complement Biology Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hughes
authors.forename: Kirsty S
authors.initials: KS
authors.lastname: Ross
authors.forename: Graeme J M
authors.initials: GJ
authors.lastname: Cowan
authors.forename: Baalasubramanian
authors.initials: B
authors.lastname: Sivasankar
authors.forename: Claire L
authors.initials: CL
authors.lastname: Harris
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mitchell
authors.forename: B Paul
authors.initials: BP
authors.lastname: Morgan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001430:Bacteriocins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D018957:CD59 Antigens / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D006461:Hemolysis / Q000502:physiology; D053878:Hemolytic Agents / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001430:Bacteriocins; D018957:CD59 Antigens; D053878:Hemolytic Agents; D010446:Peptide Fragments; C403910:intermedilysin protein, Streptococcus intermedius