Conformational Changes in C-Reactive Protein Affect Binding to Curved Membranes in a Lipid Bilayer Model of the Apoptotic Cell Surface

Abstract

C-reactive protein (CRP) is a serum protein that binds to damaged membranes through a phosphatidylcholine binding site. The membrane binding process can initiate the complement immune response and facilitates the clearance of apoptotic cells, likely aiding in the protection of autoimmunity. The initiation of an immune response relies on a conformation change from a native, pentameric form to a modified form, where the modified form binds complement proteins (i.e., C1q) and regulatory proteins substantially better than the native form. In vitro, this reactivity is observed when CRP is monomeric, and a modified form has also been observed at sites of inflammation. Despite evidence that the monomeric form has much higher affinities for almost all proteinaceous binding partners, the role of CRP conformation on lipid binding is yet unknown. In this work, we mimic the outer leaflet of apoptotic cell membranes using a nanopatterned substrate to create curved, supported lipid bilayers and then characterize how CRP conformation affects the interactions between CRP and target membranes. In this assay, the chemical composition and shape are separately tunable parameters. The lipids consisted primarily of palmitoyloleoylphosphatidylcholine, with and without lysophosphatidylcholine, and the curvature had a radius of 27-55 nm. Using this model system combined with quantitative fluorescence microscopy methods, CRP binding to lipid membranes was measured as a function of different conformations of CRP. The modified form of CRP bound curved membranes, but the pentameric form did not for the range of curvatures measured. Unlike most other curvature-sensing proteins, modified CRP accumulated more at a moderate curvature, rather than highly curved or flat regions, suggesting that the membrane bound form does not solely depend on a defect binding mechanism. The presence of lysophosphatidylcholine, a component of apoptotic membranes, increased CRP binding to all types of membranes. Overall, our results show that CRP interactions vary with protein form, lipid composition, and membrane shape. The mechanism by which CRP recognizes damaged membranes depends on the combination of all three.