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Structural basis for therapeutic inhibition of complement C5

Nature Structural & Molecular Biology volume 23, pages 378–386 (2016)Cite this article

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Abstract

Activation of complement C5 generates the potent anaphylatoxin C5a and leads to pathogen lysis, inflammation and cell damage. The therapeutic potential of C5 inhibition has been demonstrated by eculizumab, one of the world's most expensive drugs. However, the mechanism of C5 activation by C5 convertases remains elusive, thus limiting development of therapeutics. Here we identify and characterize a new protein family of tick-derived C5 inhibitors. Structures of C5 in complex with the new inhibitors, the phase I and phase II inhibitor OmCI, or an eculizumab Fab reveal three distinct binding sites on C5 that all prevent activation of C5. The positions of the inhibitor-binding sites and the ability of all three C5–inhibitor complexes to competitively inhibit the C5 convertase conflict with earlier steric-inhibition models, thus suggesting that a priming event is needed for activation.

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Figure 1: Complement inhibition by RaCI.
Figure 2: Crystal structures of C5–OmCI–RaCI complexes and binding mode of RaCI family and OmCI inhibitors.
Figure 3: Remodeling of the C5a domain in the inhibited complex.
Figure 4: The structurally conserved core of the RaCI family members is sufficient for activity.
Figure 5: Eculizumab inhibits C5 activation by binding to a site centered on the MG7 domain.
Figure 6: All the inhibited C5 complexes can themselves inhibit cleavage of C5 by the native convertase, but not all the inhibitors inhibit cleavage by a CVF convertase.
Figure 7: The structures of the inhibited C5 complexes and competition assay yield an altered view of the C5-activation pathway.

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Acknowledgements

We thank N. Darvill, T. Tang and L. Britton for experimental contributions, S. Jensen for technical support, M. Slovak (Institute of Zoology, Bratislava, Slovakia) for providing salivary glands, D.R. Greaves (University of Oxford) for mouse serum and T.E. Mollnes (Oslo University Hospital) for pig serum. We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z and Medical Research Council Hub grant G0900747 91070) for generating the sequencing data. This work was financially supported by a Rubicon grant from the Netherlands Organisation for Scientific Research to M.M.J. (825.11.030) and a Wellcome Investigator Award to S.M.L. (100298). We thank the staff at the Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy at Monash University for assistance with EM image acquisition. We acknowledge the Diamond Light Source for time on I02 under proposal MX9306. EM calculations were performed within the Multi-modal Australian Sciences Imaging and Visualisation Environment (MASSIVE).

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  1. Yang I Li & Miles A Nunn

    Present address: Present addresses: Departments of Genetics and Biology, Stanford University, Stanford, California, USA (Y.I.L.), and Akari Therapeutics, London, UK (M.A.N.).,

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Matthijs M Jore, Steven Johnson, Devon Sheppard, Natalie M Barber & Susan M Lea

  2. Department of Physiology, Medical Research Council Functional Genomics Unit, Anatomy and Genetics, University of Oxford, Oxford, UK

    Yang I Li

  3. Centre for Ecology and Hydrology, Wallingford, UK

    Miles A Nunn

  4. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia

    Hans Elmlund & Susan M Lea

  5. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, Victoria, Australia

    Hans Elmlund & Susan M Lea

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Contributions

S.M.L. and M.A.N. initially conceived the work. S.M.L. and M.M.J. designed the biochemical experiments, and M.M.J. performed them with assistance from N.M.B. M.M.J. crystallized the complexes, and the structures were solved by S.M.L. and S.J. M.M.J., S.M.L. and S.J. analyzed the structures. D.S. designed, collected and solved the NMR structure. H.E. designed the EM experiments and, together with S.M.L., collected and analyzed the data. Y.I.L. processed the HiSeq data and assembled the transcriptome. All authors were involved in discussion of results and preparation of the manuscript.

Corresponding authors

Correspondence to Hans Elmlund or Susan M Lea.

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M.M.J., M.A.N. and S.M.L. hold intellectual property in the area of use of recombinant tick complement inhibitors as therapeutic agents.

Integrated supplementary information

Supplementary Figure 1 Complement inhibition by salivary gland extract (SGE) and RaCI homologs.

(A) SGE from both male and female ticks inhibit the classical pathway in a haemolysis assay. SGE equalling 2 glands (female) or 1 gland (male) were added to the haemolysis assay. Error bars, s.e.m. (n = 3 technical replicates). (B) Inhibition of classical pathway in a haemolysis assay with supernatants from stable transfected Drosophila melanogaster S2 cell lines. Individual values of two technical replicates are shown. (C) Classical and alternative pathway inhibition by RaCI family members and OmCI using an ELISA based activation assay, similar to the Wieslab assay used in Fig. 1b. The difference between the IC50 values for CP and AP inhibition likely reflects the difference in C5 concentrations used in the assays (1% serum in CP assay versus 10% serum in the AP assay). Error bars, s.e.m. (n = 3 technical replicates). (D) Cross-species reactivity of RaCI homologues and OmCI in a haemolysis assay. Error bars, s.e.m. (n = 3 technical replicates).

Supplementary Figure 2 The custom-made Fab (EcuFab), based on the sequence of Eculizumab, is a fully active complement inhibitor.

The custom-made Fab (EcuFab), based on the sequence of Eculizumab, is a fully active complement inhibitor. Classical (CP) and alternative (AP) pathway inhibition by the Fab fragment using an ELISA based activation assay show that the IC50 values are similar to RaCIs and OmCI in Supplementary Figure 1. Error bars, s.e.m. (n = 3 technical replicates).

Supplementary Figure 3 Different location of C345c domain in our new structures of inhibited C5 compared with apo-C5.

Different location of C345c domain with respect to the rest of C5 in our new structures of inhibited C5 (wheat) compared to the earlier structures of apo-C5 (orange; Fredslund et al., Nat. Immunol. 9, 753-760, 2011). The new location is consistent with that previously seen in the CVF-C5 complex (olive; Laursen et al., EMBO J. 9,606-616, 2011). The CVF component of the C5-CVF complex is shown in a salmon ribbon representation highlighting the clash with the C345c domain in the apo-C5 like position. The residues that will form the anaphylatoxin C5a are coloured in shades of red and the overall view is the same as in the close-up of this region shown in Figure 3. The structures are overlaid by superposition of the C5a domain in each structure.

Supplementary Figure 4 Overviews of the ternary complex.

(A) Front view of C5-OmCI-RaCI3 complex. (B) Cartoon showing domain organisation of C5 and interaction sites for both tick-inhibitors (yellow stars)

Supplementary Figure 5 RaCI contacts mapped onto cross-species sequence alignment of the MG1 and MG2 domains of C5.

C5 residues that make contact with one or more RaCIs in the crystal structures are highlighted in black (van der Waals interaction) and red (salt or hydrogen bonds).

Supplementary Figure 6 RaCI contacts mapped onto cross-species sequence alignment of the C5d domain of C5.

C5 residues that make contact with one or more RaCIs in the crystal structures are highlighted in black (van der Waals interaction) and red (salt or hydrogen bonds). The large deletion in the Canis lupus sequence is likely due to sequencing or assembly errors and may not represent the actual sequence.

Supplementary Figure 7 OmCI contacts mapped onto cross-species sequence alignment of the CUB and C5d domains of C5.

C5 residues that make contact with OmCI in the crystal structures are highlighted in black (van der Waals interaction) and red (salt or hydrogen bonds). The large deletion in the Canis lupus sequence is likely due to sequencing or assembly errors and may not represent the actual sequence.

Supplementary Figure 8 OmCI contacts mapped onto cross-species sequence alignment of the C345c domain of C5.

C5 residues that make contact with OmCI in the crystal structures are highlighted in black (van der Waals interaction) and red (salt or hydrogen bonds).

Supplementary Figure 9 OmCI can bind C5 and LTB4 simultaneously.

An overlay of the structure of OmCI (blue cartoon) in complex with LTB4 (VDW spheres, carbon-green, oxygen-red) (PDB ID 3zuo; Roversi et al., J. Biol. Chem 288, 18789-18802, 2013) onto OmCI (cyan cartoon) in complex with C5 (grey cartoon) demonstrates that LTB4 binding and exchange are both compatible with C5 binding. Two views related by a rotation of 180 degrees are shown with the view on the right hand side being equivalent to the views of the complex shown in Fig. 2.

Supplementary Figure 10 The binary inhibited C5 complexes are competitive inhibitors of further C5 cleavage by the native convertase.

Purified binary C5 complexes with any of OmCI, RaCI or EcuFab compete with C5 at an initial stage of the activation pathway. –ve control is histamine-binding protein 2. Error bars, s.e.m. (n = 6; 2 independent experiments with 3 technical replicates each).

Supplementary Figure 11 Eculizumab sterically hinders the binding of CVF to C5.

Crystal structure of C5-OmCI-RaCI fitted in the EM envelope as in Figure 5E. Superposition of C5-CVF complex (Laursen et al., EMBO J. 9,606-616, 2011) on the C5-OmCI-RaCI structure results in a steric clash between CVF (salmon ribbon) and the EM volume where the Fab is positioned.

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Jore, M., Johnson, S., Sheppard, D. et al. Structural basis for therapeutic inhibition of complement C5. Nat Struct Mol Biol 23, 378–386 (2016). https://doi.org/10.1038/nsmb.3196

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