Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the...

Area of Science:

• Immunology
• Biochemistry
• Surface Science

Background:

• The complement system regulates host and foreign surface interactions.
• Mechanisms of complement activation and regulation by surface molecules are not fully understood.
• Complement system proteins C3b and factor H (FH) are key players in complement regulation.

Purpose of the Study:

• To investigate the single-molecule interactions between C3b and FH.
• To understand the role of surface chemistry in FH-C3b complex formation and stability.
• To explore the implications for complement-mediated diseases like atypical hemolytic uremic syndrome.

Main Methods:

• Combined Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM) for single-molecule analysis.
• SPR monitored complement initiation via a C3b-mediated positive-feedback loop.
• AFM force-distance measurements assessed FH detachment from C3b complexes.

Main Results:

• C3b deposition was amplified on the SPR chip, forming clusters and distant solitary molecules.
• A force of 0.17 ± 0.02 nN was required to detach FH from C3b, with variable stretching.
• A disease-associated FH variant (FH(D1119G)) detached more uniformly and easily.
• FH-C3b binding affinity (K values) was 5-fold tighter on a custom surface compared to commercial chips, mimicking erythrocyte surfaces.

Conclusions:

• Surface chemistry critically influences the binding affinity and conformational dynamics of FH-C3b interactions.
• These findings highlight the functional importance of surface-driven conformational changes in complement regulation.
• Understanding these interactions provides insights into complement-related pathologies and potential therapeutic targets.