Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.

T 细胞和吞噬细胞等免疫监视细胞利用完整的质膜受体来识别触发和激活细胞（例如凋亡细胞）的表面特征。跨膜免疫球蛋白和粘蛋白结构域 (Tim) 蛋白就是这样的质膜传感器家族之一，可特异性识别磷脂酰丝氨酸 (PS)，但会引发不同的免疫反应。识别靶细胞表面脂质信号的分子基础尚不清楚。先前的结果表明，Tim 蛋白膜界面上存在的碱性侧链可能有助于与其他阴离子脂质（包括但不一定限于 PS）结合。因此，我们在生理相关条件下对小鼠 Tim1、Tim3 和 Tim4 与合成阴离子磷脂膜的结合进行了比较定量分析。 X 射线反射率和囊泡结合研究用于将 Tim3 的水溶性结构域与之前获得的 Tim1 和 Tim4 的结果进行比较。尽管钙连接对于所有三种蛋白质都是必需的，但这三种同源物在平衡驱动膜缔合的疏水性和静电相互作用的方式上有所不同。这些蛋白质对磷脂链不饱和度的感知也有所不同，并且在对双层 PS 浓度的依赖性方面表现出不同程度的协同性。令人惊讶的是，微量阴离子磷脂酸极大地增强了 Tim3 和 Tim4 的双层缔合，但没有增强 Tim1 的缔合。一种新颖的数学模型提供了结合参数的值，并阐明了配体之间复杂的相互作用。 总之，我们的结果提供了三种 Tim 蛋白在识别靶细胞表面磷脂时使用的对比选择性的定量描述。该范例通常适用于通过多个配体的异向协作相互作用来分析外周蛋白与靶膜的结合。