Binding of the lipocalin C8γ to human complement protein C8α is mediated by loops located at the entrance to the C8γ ligand binding site

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the membrane attack complex (MAC). C8 is composed of a disulfide-linked C8α-γ heterodimer and a noncovalently associated C8β chain. C8α and C8β are homologous to C6, C7 and C9, whereas C8γ is the only lipocalin in the complement system. Lipocalins have a core β-barrel structure forming a calyx with a binding site for a small molecule. In C8γ, the calyx opening is surrounded by four loops that connect β-strands. Loop 1 is the largest and contains Cys40 that links to Cys164 in C8α. To determine if these loops mediate binding of C8α prior to interchain disulfide bond formation in C8α-γ, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8γ. The siderocalin-C8γ chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8α. Results indicate at least three of the four loops surrounding the entrance to the C8γ calyx are involved in binding C8α. Binding near the calyx entrance suggests C8α may restrict and possibly regulate access to the C8γ ligand binding site.