Chiswell Brian, Slade Daniel J, Sodetz James M
Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Columbia, 29208, USA.
Biochim Biophys Acta. 2006 Sep;1764(9):1518-24. doi: 10.1016/j.bbapap.2006.07.003. Epub 2006 Jul 28.
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 C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous to C6, C7 and C9, whereas C8gamma 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 C8gamma, 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 C8alpha. To determine if these loops mediate binding of C8alpha prior to interchain disulfide bond formation in C8alpha-gamma, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8gamma. The siderocalin-C8gamma chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8alpha. Results indicate at least three of the four loops surrounding the entrance to the C8gamma calyx are involved in binding C8alpha. Binding near the calyx entrance suggests C8alpha may restrict and possibly regulate access to the C8gamma ligand binding site.
人C8是相互作用形成膜攻击复合物（MAC）的五种补体成分（C5b、C6、C7、C8和C9）之一。C8由一个二硫键连接的C8α-γ异二聚体和一个非共价结合的C8β链组成。C8α和C8β与C6、C7和C9同源，而C8γ是补体系统中唯一的脂质运载蛋白。脂质运载蛋白具有一个核心β桶结构，形成一个带有小分子结合位点的杯状结构。在C8γ中，杯状结构的开口被连接β链的四个环包围。环1最大，包含与C8α中的Cys164相连的Cys40。为了确定这些环是否在C8α-γ链间二硫键形成之前介导C8α的结合，将这些环分别或组合替换为与C8γ结构相似的脂质运载蛋白——铁调素（NGAL，Lcn2）中的相应环。铁调素-C8γ嵌合构建体在大肠杆菌中表达、纯化，并检测其结合C8α的能力。结果表明，围绕C8γ杯状结构入口的四个环中至少有三个参与结合C8α。在杯状结构入口附近的结合表明C8α可能限制并可能调节对C8γ配体结合位点的访问。
