Drücker Patrick, Pejic Milena, Grill David, Galla Hans-Joachim, Gerke Volker
Institute of Biochemistry, University of Muenster, Muenster, Germany.
Institute of Medical Biochemistry, ZMBE, University of Muenster, Muenster, Germany.
Biophys J. 2014 Nov 4;107(9):2070-81. doi: 10.1016/j.bpj.2014.08.027.
Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca(2+)-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with Kd values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.
生物膜被组织成动态微结构域，这些微结构域是信号转导和膜运输的场所。这些微结构域的形成和扩展由膜脂以及整合蛋白和膜相关蛋白的内在特性驱动。膜联蛋白A2（AnxA2）是一种外周相关膜蛋白，它可以以Ca(2+)依赖的方式支持微结构域的形成，并参与膜运输过程。在这里，我们对AnxA2与含有不同组成的膜联蛋白结合脂质磷脂酰肌醇-4,5-二磷酸和磷脂酰丝氨酸的固体支持膜的结合进行了定量分析。我们表明，这种结合具有高特异性和亲和力，解离常数在22.1至32.2 nM之间。我们还分析了AnxA2与其S100A10蛋白配体的异源四聚体复合物的结合参数，结果表明该复合物对相同膜具有更高的亲和力，Kd值为12至16.4 nM。有趣的是，单体AnxA2和AnxA2-S100A10复合物的结合具有正协同性。这种协同结合由该蛋白保守的C末端膜联蛋白核心结构域介导，并且需要胆固醇的存在。据我们所知，我们的结果首次揭示AnxA2及其衍生物与含有胆固醇、磷脂酰丝氨酸和/或磷脂酰肌醇-4,5-二磷酸的膜协同结合，从而为AnxA2的脂质聚集活性提供了一个机制模型。
