Spider-like proteins spin defenses to control immunity

Human blood contains spider-shaped proteins that regulate the innate immune response. When antibodies attach to a pathogen, complement protein C1 binds to them and triggers a cascade of protein cleavages that amplify the immune signal. C1 activation initiates successive complement protein cleavages and amplifies the signal to the immune system.

To prevent harmful inflammation, the body relies on regulators to shut down the cascade. Researchers from Utrecht University, including Tereza Kadavá and Albert Heck, Leiden University Medical Center and the University of Applied Sciences in Austria revealed how the spider-like complement regulator C4b-binding protein, or C4BP, binds its immune targets. They reported their results in Molecular & Cellular Proteomics.

Albert Heck

This 3D visualization of C4BP was created by Hecklab in collaboration with Sensu.health. C4BP uses its eight “legs” to catch proteins related to inflammation, stopping further immune response.

C4b plays a role in the classical and lectin complement pathways, which culminate in the formation of the membrane attack complex, or MAC. The MAC is a cylindrical protein complex that forms pores in the pathogenic bacterial cell membrane, which leads to cell death.

C4BP is an essential cofactor for serine protease factor I, or FI, which cleaves C4b and generates inactive C4b, or iC4b. This blocks activation of the rest of the complement cascade and prevents MAC formation.

Previous studies have found that most human serum C4BP is bound to serum amyloid P component, or SAP. When not bound to C4BP, SAP can bind to viruses, bacteria and malaria parasites and induce phagocytosis of the bound pathogens.

While it is known that C4BP, C4b and SAP play a role in complement inhibition, the team wanted to better understand the C4BP-C4b and C4BP-SAP complex structures. They used mass photometry, cross-linking mass spectrometry and high-speed atomic force microscopy to image the unbound proteins and the complexes they form. Mass photometry is a technique that uses a laser to detect single molecules in solution as they adsorb onto a glass surface.

They found that the N-terminal domain of a C4BPα chain, or one leg of the spider-like C4BP, binds to C4b in a 1:1 ratio. This allows the C4BP higher-order structure, or HOS, to bind multiple molecules of C4b.

When bound with SAP, researchers found a strong preference for 1:1 C4BP:SAP ratios. SAP did not bind to a single C4BPα construct, only the intact full C4BP HOS. AFM Microscopy revealed C4BPα chains encircling the SAP protein, with the C4BP core perched on top, like a spider grasping a fly.

These findings support previous research on the regulatory mechanisms of C4BP-SAP complex formation. C4b and SAP can both bind to C4BP, but SAP likely limits the flexibility of C4BPα chains, reducing its ability to inhibit the complement system.

SAP is a therapeutic target for treating fibrosing diseases associated with chronic inflammation. Previous studies proposed that human serum C4BP is mostly bound to SAP when the immune complement system is not triggered. This study determined the binding stoichiometry between C4BP and SAP as well as the structural configuration, which could aid drug development targeting SAP.