Eculizumab is a humanized mAb approved for treatment of patients with paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab binds complement component C5 and prevents its cleavage by C5 convertases, inhibiting release of both the proinflammatory metabolite C5a and formation of the membrane attack complex via C5b. In this study, we present the crystal structure of the complex between C5 and a Fab fragment with the same sequence as eculizumab at a resolution of 4.2 Å. Five CDRs contact the C5 macroglobulin 7 domain, which contains the entire epitope. A complete mutational scan of the 66 CDR residues identified 28 residues as important for the C5–eculizumab interaction, and the structure of the complex offered an explanation for the reduced C5 binding observed for these mutant Abs. Furthermore, the structural observations of the interaction are supported by the reduced ability of a subset of these mutated Abs to inhibit membrane attack complex formation as tested in a hemolysis assay. Our results suggest that eculizumab functions by sterically preventing C5 from binding to convertases and explain the exquisite selectivity of eculizumab for human C5 and how polymorphisms in C5 cause eculizumab-resistance in a small number of patients with paroxysmal nocturnal hemoglobinuria.

The structure of the human C5aR antagonist, C5a-A8, reveals a three-helix bundle conformation similar to that observed for human C5a-desArg, whereas murine C5a and C5a-desArg both form the canonical four-helix bundle. These conformational differences are discussed in light of the differential C5aR activation properties observed for the human and murine complement anaphylatoxins across species.

The complement system is a highly complex and carefully regulated proteolytic cascade activated through three different pathways depending on the activator recognized. The structural knowledge regarding the intricate proteolytic enzymes that activate and control complement has increased dramatically over the last decade. This development has been pivotal for understanding how mutations within complement proteins might contribute to pathogenesis and has spurred new strategies for development of complement therapeutics. Here we describe and discuss the complement system from a structural perspective and integrate the most recent findings obtained by crystallography, small-angle X-ray scattering, and electron microscopy. In particular, we focus on the proteolytic enzymes governing activation and their products carrying the biological effector functions. Additionally, we present the structural basis for some of the best known complement inhibitors. The large number of accumulated molecular structures enables us to visualize the relative size, position, and overall orientation of many of the most interesting complement proteins and assembled complexes on activator surfaces and in membranes.

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