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 C8 alpha-gamma heterodimer and a noncovalently associated C8 beta chain. C8 alpha and C8 beta are homologous to C6, C7 and C9, whereas C8 gamma is the only lipocalin in the complement system. Lipocalins have a core beta-barrel structure forming a calyx with a binding site for a small molecule. In C8 gamma, the calyx opening is surrounded by four loops that connect beta-strands. Loop 1 is the largest and contains Cys40 that links to Cys164 in 8 alpha. To determine if these loops mediate binding of C8 alpha prior to interchain disulfide bond formation in C8 alpha-gamma, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8 gamma. The siderocalin-C8 gamma chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8 alpha. Results indicate at least three of the four loops surrounding the entrance to the C8 gamma calyx are involved in binding C8a. Binding near the calyx entrance suggests C8 alpha may restrict and possibly regulate access to the C8 gamma ligand binding site. (c) 2006 Elsevier B.V. All rights reserved.