Structural Insights into Complement Inhibition: Visualizing Distinct Binding Modes of C4b-binding Protein Complexes with C4b and SAP

C4b-binding protein (C4BP) is an innate immune inhibitor found in serum. Human C4BP adopts spider-like higher-order structures (HOS) formed by disulfide-linked C4BPα and C4BPβ chains that non-covalently bind vitamin K-dependent protein S (ProS). These spider-like structures can form even larger complexes as C4BP interacts with other, mostly complement-related, proteins. The complement inhibitory role of C4BP is primarily mediated through its interaction with C4b. C4BP also binds with high affinity to serum amyloid P component (SAP), a pentraxin family member associated with amyloidosis conditions. Here, we structurally and compositionally characterize C4BP interactions with these two natively occurring binders. To achieve this, we combine mass photometry, high-speed atomic force microscopy, and cross-linking mass spectrometry. By integrating the results, we reveal two distinct binding modes of C4BP when bound to C4b or SAP. Given the spider-like assembly of C4BP, C4b interacts with the N-terminal region of a single C4BPα leg, enabling multiple C4b molecules to bind to the C4BP HOS. Conversely, SAP engages with the entire spider-like HOS: the C4BPα-C4BPβ oligomerization core binds to SAP, and the C4BPα legs wrap around it.