Crystal structure of complement protein C8γ in complex with a peptide containing the C8γ binding site on C8α: Implications for C8γ ligand binding

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex. It contains three genetically distinct subunits; C8α and C8γ form a disulfide-linked C8α-γ heterodimer that is noncovalently associated with C8β. The C8α subunit is homologous to C8β, C6, C7 and C9 and together they form the MAC family of proteins. By contrast, C8γ is the only lipocalin in the complement system. Like other lipocalins, it has a core β-barrel structure forming a calyx with a distinct binding pocket for a small and as yet unidentified ligand. The binding site on C8α for C8γ was previously localized to a 19-residue segment which contains an insertion (indel) that is unique to C8α. Included in the indel is C8α Cys 164 which links to Cys 40 in C8γ. In the present study, C8γ containing a C40A substitution was co-crystallized with a synthetic indel peptide containing the equivalent of a C8α C164A substitution. The X-ray crystal structure shows that the indel peptide completely fills the upper portion of the putative C8γ ligand binding pocket and is in contact with all four loops at the calyx entrance. The lower part of the C8γ cavity is either unoccupied or contains disordered solvent. The validity of the structure is supported by the spatial arrangement of C8α Ala 164 in the peptide and C8γ Ala 40, which are within disulfide-bonding distance of each other. Corresponding studies in solution indicate the C8γ ligand binding site is also occupied by the indel segment of C8α in whole C8. These results suggest a role for C8α in regulating access to the putative C8γ ligand binding site.