Abstract
The abnormal formation of the membrane attack complex (MAC) is intrinsically linked to a range of acute and chronic immune diseases. The insertion of complement C9 into the membrane is the final step and kinetic bottleneck of MAC formation. However, research on blocking the MAC formation of C9 is currently limited. Given its broad, flat, and polar functional interface, complement C9 is a challenging target for rational design. Here, we utilize deep learning-based methods for protein scaffold generation, sequence design, and complex structure prediction to de novo design mini-protein inhibitors that specifically block the membrane insertion of soluble complement C9. The binding affinity of the mini-protein inhibitor is further optimized to 700 pM via partial diffusion. Design accuracy and binding specificity are verified through X-ray crystallography and biochemical studies. An in vivo acute hemolysis inhibition assay reveals that the C9 mini-protein inhibitors remain effective against hemolysis even 8 minutes after complement activation, outperforming the complement C5 inhibitor eculizumab. The de novo designed C9 mini-protein inhibitors can offer an optional therapeutic approach for the prevention and treatment of acute or chronic immune diseases associated with abnormal complement activation.
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Data availability
Coordinates and structure files have been deposited to the Protein Data Bank with accession codes 9X1W (P57-M4), 9X1X (P57-M5). Full raw data including all tested models are available on zenodo.org (accession code: 18530064). The final sequences of the designed inhibitors optimized by partial diffusion are provided in Supplementary Data 1. Source data is available with this paper as a Source Data file. Source data are provided with this paper.
Code availability
The code used to generate the scaffolds of mini-protein binders in this study has been previously published35 and is publicly available on GitHub at https://github.com/RosettaCommons/RFdiffusion, under BSD 3-Clause License. The code used to sequence design and complex structure generation has been previously published56 and is publicly available on GitHub at https://github.com/nrbennet/dl_binder_design, under MIT License (for the main codebase) and Apache-2.0 License (for AlphaFold2 module). The code we developed has been uploaded to Zenodo with https://doi.org/10.5281/zenodo.18530064, under BSD 3-Clause License.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (32501303 to B.Y., 82303251 to N.W., 81873883 to S.L., 82000525 to M.F.L.), the Shandong Provincial Natural Science Foundation, China (ZR2022QC209 to B.Y., ZR2023QH202 to N.W.). This work was also supported by the Shandong Provincial Science and Technology Support Plan for Youth Innovation in Universities (10438202502 to B.Y.) and the Graduate Student Research Grant from Shandong Second Medical University (2025YJSCX022). We would like to thank the Protein Characterization and Crystallography Facility of Westlake University for help in sample analysis; the Mass Spectrometry & Metabolomics Core Facility of Westlake University for sample analysis; and the Westlake University HPC Center for computation assistance.
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B.Y. and S.L. designed the research. B.Y. made the designs. M.L., N.W., X.F., G.W., and Z.Z. performed most of the experiments with the help of Y.Y., T.X., Y.Z., J.P., D.W., M.F.L., Y.L., J.T., Z.J. All authors analyzed data. B.Y., S.L,. and L.C. supervised research. B.Y., S.L., and M.L. wrote the manuscript with the input from the other authors. All authors revised the manuscript.
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B.Y., S.L., M.L., X.F., and N.W. are coinventors on a patent application for invention (applicant: Shandong Second Medical University; application number: CN202511704580.7; status: under substantive examination) that incorporates the usage of mini-protein binders P9 and P57 in hemolysis inhibition. The remaining authors declare no competing interests.
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Li, M., Wang, N., Fu, X. et al. Design of miniprotein inhibitors targeting complement C9 to block membrane attack complex assembly. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70667-x
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DOI: https://doi.org/10.1038/s41467-026-70667-x
