The Complement System
The complement system is an evolutionary ancient immune defense mechanism, present in vertebrates and invertebrates.It forms a first line of defense in plasma and fluids surrounding tissues and enables the host to recognise and clear invading pathogens (bacteria and virusses) and alters host cells, while proctecting healthy host cells and tissues. In humans this system is formed by more than 30 large modular plasma proteins and cell surface receptors.
Antibody mediated activation of complement
The classical pathway of complement is activated when C1 binds to surface-bound antibody complexes. Antibody-mediated complement activation may lead to cell lysis (CDC) or lead to 'silent' (non-inflammatory) apoptosis of host cells. By studying the molecular processes of complex formation, we hope to address questions, such as what determines the CDC of an antibody and how does IgM stimulate ‘silent’ removal of apoptotic cells?
We have shown that IgG antibodies form hexameric arrangements that bind C1 and activate C1. Specific mutations at the Fc-Fc interface that stabilize the hexamers enhance antibody-mediated complement activation against tumour models in vitro. We used cryo-EM single particle analysis to study stabilized C1-IgG16 complexes and cryo-EM tomography to image the first steps of IgM-mediated complement activation.
The antibodies adopt hexagonal arrangements that bind the C1q globular heads.of the C1 complex. The data suggests that the C1q collagen arms are pulled together upon antobody binding, with different antibody-types giving a variation in the spreadig of the six C1q-collagen helices. We postulated a mechanism for surface activation of C1 that depends on the hexameric arrangement of C1 binding platforms. Binding of C1q to these platforms skews the hexameric arrangement of the collagen arms that bind the protease platform which activates C1 for cleavage of C4.
The Alternative pathway
We have resolved the structural details and elucidated the molecular mechanisms of the central components of the complement system that lead to amplification of the complement response and is referred to as the Alternative Pathway of complement activation. We determined three-dimensional structures of the central complements of component in various activation states,
Collectively these structures show that factor B binds through two interfaces, one formed by the CCP domains of the pro-peptide segment Ba and one through the VWA domain of the protease segment Bb. Binding of factor B to C3b induces a dynamic equilibrium between a closed (loading) and open (activation) form. Factor D circulates in blood in an inactive confomation and binds the open form of the pro-convertase C3bB. Binding activates factor D by inducing rearrangement in the self-inhibitory loop that restore the catalytic triad. Factor D then cleaves factor B, releasing the anaphylatoxin fragment Ba and yielding the active C3 convertase, C3bBb.
The C3 convertase is a very unstable short-lived complexe, but we were able to determine its 3-dimensional structure using an immune evasion protein of Staphylococcus aureus, called SCIN, that inhibits and stabilises C3 convertases. The structure revealed a dimeric arrangement of convertases stabilised by two bridging SCIN molecules. The observed C3b:C3b contacts may partially mimic the enzyme:substrate contacts of the convertase. This model is now corroborated by findings from other groups.
Complement regulators
There is just one known positive regulator of complement that occurs in blood as flexible dimers, trimers and tetramers. Properdin enhances C3b deposition on surfaces by stabilising surface-bound alternative pathway C3 convertases. We solved the structure of this properdin and its complex with the CTC domain of C3b by co-expressing properdin as separate N- and C-terminal constructs that form a single C3b-binding properdin "monomer" and proposed a model that explaines how oligomerisation of properdin directs the activity of properdin towards C3b-deposited surfaces.
Surface binding of C3b in indiscriminate between self and foreign surfaces. Therefore host cells and tissues are protected by a plethora of negative regulators of complement. These act by either destabilising the convertase (decay-acceleration), or form a binding platform for the protease factor I, which cleaves and inactivates C3b (co-factor activity). Some regulators, like factor H, have both activities. To escape complement activation, several pathogens use mimmics of these negative regulators.. We solved the crystal structures of 4 human (FH, MCP, CR1 and DAF) and one viral (SPICE) complement regulator in complex with C3b as well as the structure of the tertiary complex of Factor I, Factor H and C3b.
The structures show a very similar binding platform for all regulators and explain the differences in activity between the various regulators
Membrane-Attack complex
Activation of the terminal pathway of complement results in formation of membrane-attack complexes that from large (100 Å wide) pores in the target membrane.These complexes are formed when C5 is cleaved into C5b by the C5 convertase. C5b binds subsequently C6, C7, (trimeric) C8αβγ and multiple copies of C9. We determined the structure of the membrane-insertion (MACPF) domain of complement component C8α and the C5b-C6 (C5b6) complex. In collaboration with the labs of Susan Lea (Oxford) and Oscar Llorca (Madrid) we placed the C5b6 crystal structure into the cryo-EM reconstruction map of the soluble MAC (sMAC of sC5b6-9). These data indicate the arrangement of C5b-C6-C7-C8β-C8αγ-C9, which forms an arc of the MAC proteins with a protrusion at the beginning formed by C5b.
Phase-plate cryo-EM tomographic series revealed extensive structural heterogeneities in MAC formation.Complexes of C5b-7 adhere to bilayers, bulging out the outer-leaflet, while maintaining the integrity of the membrane inner-leaflet. In contrast, oligomeric structures of C5b-8 perforate the bilayer, forming a oligomeric C5b-8 pore, explaining that membrane leakage is observed in the absence of the ring-forming C9. C5b-9 (MAC) rings were observed forming both single pores and multimeric pores of various shapes.