An indel within the C8 alpha subunit of human complement C8 mediates intracellular binding of C8 gamma and formation of C8 alpha-gamma.
Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.
Plumb
M E
ME
Department of Chemistry and Biochemistry and the School of Medicine, University of South Carolina, Columbia, South Carolina 29208, USA.
Sodetz
J M
JM
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003185:Complement C8 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D015941:Complement Hemolytic Activity Assay; D015938:Complement Membrane Attack Complex / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011110:Polymorphism, Genetic; D011485:Protein Binding / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D003185:Complement C8; D015938:Complement Membrane Attack Complex; D010446:Peptide Fragments; D003545:Cysteine