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Dale L. Bodian^*, Simon J. Davis, B. Paul Morgan, and Neil K. Rushmere

From the ^* Laboratory of Molecular Biophysics, Oxford OX1 3QU, United Kingdom; Molecular Sciences Division, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom; and Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff CF4 4XN, United Kingdom

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extracellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

The authors would like to thank A.N. Barclay for his generous support of the initial stages of these experiments, L.M. Baggott and F.M. Flavin for assistance with sequencing and P.C. Driscoll for providing NMR coordinates of human CD59. D.L. Bodian was supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation, Fellowship DRG-1246. S.J. Davis, N.K. Rushmere and B.P. Morgan are funded by the Wellcome Trust.

¹ Abbreviations used in this paper: CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; Ly-6SF, Ly-6 superfamily; MAC, membrane attack complex; MIRL, membrane inhibitor of reactive lysis; NHS, normal human serum; NMR, nuclear magnetic resonance; RT, room temperature.

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