Sites within the complement C3b/C4b receptor important for the specificity of ligand binding.
Cysteine-rich repeated units of 40-70 amino acids are building blocks of many mammalian proteins, including 12 proteins of the complement system. Human complement arranged motifs, designated short consensus repeats (SCRs), which constitute the entire extracellular portion of this protein. Klickstein et al. [Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A. & Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717 (abstr.)] localized a C4b binding domain to SCR-1 and/or SCR-2 and a C3b binding domain to SCR-8 and/or SCR-9. These SCRs bind different ligands, although SCR-1 and SCR-8 are 55% homologous and SCR-2 and SCR-9 are 70% homologous. To examine if one or two SCRs are required for ligand binding and to define sites within the SCRs that determine specificity of binding, mutagenesis analysis of a truncated, secreted form of CR1, called CR1-4 by Hourcade et al. [Hourcade, D., Meisner, D. R., Atkinson, J. P. & Holers, V. M. (1988) J. Exp. Med. 168, 1255-1270], was undertaken. The latter, composed of the first eight and one-half amino-terminal SCRs of CR1, efficiently bound C4b but not iC3. SCR-1 and SCR-2 were necessary for this interaction. Analysis of the mutant CR1-4 proteins, in which amino acids in SCR-1 and SCR-2 were substituted a few at a time with the homologous amino acids of SCR-8 and SCR-9, led to the identification of one amino acid in SCR-1 and three amino acids in SCR-2 important for C4b binding. Furthermore, five amino acids at the end of SCR-9, if placed in the homologous positions of SCR-2, conferred iC3 binding and are likely essential for ligand binding activity of SCR-8 and SCR-9. This iC3 binding occurred only if SCR-1 was present, indicating that two contiguous SCRs are necessary for this interaction. These results provide identification of amino acids within SCRs that are important for ligand binding.
Krych
M
M
Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.
Hourcade
D
D
Atkinson
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003179:Complement C3b / Q000378:metabolism; D015935:Complement C4b / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D051381:Rats; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics; D017463:Receptors, Complement 3b; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid
D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D003179:Complement C3b; D015935:Complement C4b