Zhang X, Rizo J, Südhof T C
Department of Molecular Genetics, Center for Basic Neuroscience, Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas 75235, USA.
Biochemistry. 1998 Sep 8;37(36):12395-403. doi: 10.1021/bi9807512.
Synaptotagmin I is a synaptic vesicle membrane protein that probably functions as a Ca2+ sensor in neurotransmitter release and contains two C2-domains which bind Ca2+. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids in a Ca2+-dependent manner similar to that of the C2-domains of protein kinase C, cytoplasmic phospholipase A2, and phospholipase Cdelta1. Although the tertiary structure of these C2-domains is known, the molecular basis for their Ca2+-dependent interactions with phospholipids is unclear. We have now investigated the mechanisms involved in Ca2+-dependent phospholipid binding by the C2A-domain of synaptotagmin I. Our data show that the C2A-domain binds negatively charged liposomes in an electrostatic interaction that is determined by the charge density of the liposome surface but not by the phospholipid headgroup. At the tip of the C2A-domain, three tightly clustered Ca2+-binding sites are formed by five aspartates and one serine. Mutations in these aspartate and serine residues demonstrated that all three Ca2+-binding sites are required for phospholipid binding. The Ca2+ binding sites at the top of the C2A-domain are surrounded by positively charged amino acids that were shown by mutagenesis to be also involved in phospholipid binding. Our results yield a molecular picture of the interactions between a C2-domain and phospholipids. Binding is highly electrostatic and occurs between the surfaces of the phospholipid bilayer and of the tip of the C2A-domain. The data suggest that the negatively charged phospholipid headgroups interact with the basic side chains surrounding the Ca2+-binding sites and with bound Ca2+ ions, thereby filling empty coordination sites and increasing the apparent affinity for Ca2+. In addition, insertion of hydrophobic side chains may contribute to phospholipid binding. This model is likely to be general for other C2-domains, with the relative contributions of electrostatic and hydrophobic interactions dictated by the exposed side chains surrounding the Ca2+-binding region.
突触结合蛋白I是一种突触小泡膜蛋白，可能在神经递质释放过程中作为Ca2+传感器发挥作用，它含有两个结合Ca2+的C2结构域。突触结合蛋白I的第一个C2结构域（C2A结构域）以与蛋白激酶C、细胞质磷脂酶A2和磷脂酶Cδ1的C2结构域类似的Ca2+依赖性方式结合磷脂。尽管这些C2结构域的三级结构已知，但其与磷脂的Ca2+依赖性相互作用的分子基础尚不清楚。我们现在研究了突触结合蛋白I的C2A结构域与Ca2+依赖性磷脂结合所涉及的机制。我们的数据表明，C2A结构域通过静电相互作用结合带负电荷的脂质体，这种相互作用由脂质体表面的电荷密度决定，而不是由磷脂头部基团决定。在C2A结构域的顶端，由五个天冬氨酸和一个丝氨酸形成了三个紧密聚集的Ca2+结合位点。这些天冬氨酸和丝氨酸残基的突变表明，所有三个Ca2+结合位点都是磷脂结合所必需的。C2A结构域顶部的Ca2+结合位点被带正电荷的氨基酸包围，诱变显示这些氨基酸也参与磷脂结合。我们的结果给出了一个C2结构域与磷脂之间相互作用的分子图景。结合是高度静电性的，发生在磷脂双层表面和C2A结构域顶端之间。数据表明，带负电荷的磷脂头部基团与Ca2+结合位点周围的碱性侧链以及结合的Ca2+离子相互作用，从而填充空的配位位点并增加对Ca2+的表观亲和力。此外，疏水侧链的插入可能有助于磷脂结合。这个模型可能适用于其他C2结构域，静电和疏水相互作用的相对贡献由Ca2+结合区域周围暴露的侧链决定。
