Alnaas Aml A, Moon Carrie L, Alton Mitchell, Reed Scott M, Knowles Michelle K
Department of Chemistry and Biochemistry, University of Denver , Denver, Colorado 80208, United States.
Molecular and Cellular Biophysics, University of Denver , Denver, Colorado 80208, United States.
J Phys Chem B. 2017 Mar 30;121(12):2631-2639. doi: 10.1021/acs.jpcb.6b11505. Epub 2017 Mar 15.
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.
C反应蛋白（CRP）是一种血清蛋白，它通过磷脂酰胆碱结合位点与受损细胞膜结合。膜结合过程可启动补体免疫反应并促进凋亡细胞的清除，可能有助于保护机体免受自身免疫的影响。免疫反应的启动依赖于从天然五聚体形式到修饰形式的构象变化，修饰形式比天然形式能更好地结合补体蛋白（即C1q）和调节蛋白。在体外，当CRP呈单体形式时可观察到这种反应性，并且在炎症部位也观察到了修饰形式。尽管有证据表明单体形式对几乎所有蛋白质结合伙伴具有更高的亲和力，但CRP构象对脂质结合的作用尚不清楚。在这项研究中，我们使用纳米图案化底物模拟凋亡细胞膜的外小叶，以创建弯曲的、支撑的脂质双层，然后表征CRP构象如何影响CRP与靶膜之间 的相互作用。在该测定中，化学成分和形状是可分别调节的参数。脂质主要由棕榈酰油酰磷脂酰胆碱组成，有或没有溶血磷脂酰胆碱，曲率半径为27 - 55纳米。使用该模型系统结合定量荧光显微镜方法，测量了CRP与脂质膜的结合情况，作为CRP不同构象的函数。CRP的修饰形式结合弯曲膜，但在测量的曲率范围内，五聚体形式不结合。与大多数其他曲率传感蛋白不同，修饰后的CRP在中等曲率处积累更多，而不是在高度弯曲或平坦区域，这表明膜结合形式不仅仅取决于缺陷结合机制。凋亡细胞膜的成分溶血磷脂酰胆碱的存在增加了CRP与所有类型膜的结合。总体而言，我们的结果表明CRP的相互作用随蛋白质形式、脂质组成和膜形状而变化。CRP识别受损膜的机制取决于这三者的组合。
