The complement pathway is one of the most ancient elements of the host's innate response and includes a set of protein effectors that rapidly react against pathogens. The late stages of the complement reaction are broadly categorised into two major outcomes. Firstly, C5a receptors, expressed on membranes of host cells, are activated by C5a to generate pro-inflammatory responses. Secondly, target cells are lysed by a hetero-oligomeric pore known as the membrane attack complex (MAC) that punctures the cellular membrane, causing ion and osmotic flux. Generally, several membrane-bound and soluble inhibitors protect the host membrane from complement damage. This includes inhibitors against the MAC, such as clusterin and CD59. This review addresses the most recent molecular and structural insights behind the activation and modulation of the integral membrane proteins, the C5a receptors (C5aR1 and C5aR2), as well as the regulation of MAC assembly. The second aspect of the review focuses on the molecular basis behind inflammatory diseases that are reflective of failure to regulate the terminal complement effectors. Although each arm is unique in its function, both pathways may share similar outcomes in these diseases. As such, the review outlines potential synergy and crosstalk between C5a receptor activation and MAC-mediated cellular responses. Graphical Abstract

Iron is an essential nutrient for most bacteria and is often growth-limiting during infection, due to the host sequestering free iron as part of the innate immune response. To obtain the iron required for growth, many bacterial pathogens encode transporters capable of extracting the iron-containing cofactor heme directly from host proteins. Pathogenic E. coli and Shigella spp. produce the outer membrane transporter ChuA, which binds host hemoglobin and extracts its heme cofactor, before importing heme into the cell. Heme extraction by ChuA is a dynamic process, with the transporter capable of rapidly extracting heme from hemoglobin in the absence of an external energy source, without forming a stable ChuA-hemoglobin complex. In this work, we utilise a combination of structural modelling, Cryo-EM, X-ray crystallography, mutagenesis, and phenotypic analysis to understand the mechanistic detail of this process. Based on this understanding we utilise artificial intelligence-based protein design to create binders capable of inhibiting E. coli growth by blocking hemoglobin binding to ChuA. By screening a limited number of these designs, we identify several binders that inhibit E. coli growth at low nanomolar concentrations, without experimental optimisation. We determine the structure of a subset of these binders, alone and in complex with ChuA, demonstrating that they closely match the computational design. This work demonstrates the utility of de novo-designed proteins for inhibiting bacterial nutrient uptake and uses a workflow that could be applied to integral membrane proteins in other organisms.

The Membrane Attack Complex (MAC) is responsible for forming large β-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

The terminal C5b-9 complement complex (TCC) exists in two forms; the soluble sC5b-9 and the solid-phase inserted Membrane Attack Complex (MAC). The MAC is responsible for forming large β-barrel channels in the membranes of pathogens and can target cells such as gram-negative bacteria. In addition, off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex and in polyC9, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MACrelated inflammation, and pathophysiology. However, the identity of this C9 neoepitope remains unknown. Herein, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Åresolution. The aE11 binding site revealed that the neoepitope was formed by two separate surfaces on the pore periphery, rather than a conformational change in the protein structure, and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, our observations indicated that aE11 can recognise a partial epitope in purified monomeric C9, albeit binding was two orders of magnitude weaker. Taken together, our results reveal the basis for MAC recognition by aE11 and that the C9 neoepitope can be formed without substantial conformational rearrangements adding insight into how aE11 is used to quantify MAC formation in disease.