Shao X, Fernandez I, Südhof T C, Rizo J
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Biochemistry. 1998 Nov 17;37(46):16106-15. doi: 10.1021/bi981789h.
C2 domains are widespread Ca2+-binding modules that are particularly abundant in proteins involved in membrane traffic and signal transduction. The C2A domain of synaptotagmin I is believed to play a key role in neurotransmitter release through its Ca2+-dependent interactions with syntaxin and phospholipids. Elucidating the structural consequences of Ca2+ binding to the C2A domain is critical for understanding its mechanism of action and for models of the functions of other C2 domains. We have determined the solution structure of the Ca2+-free and Ca2+-bound forms of the C2A domain of synaptotagmin I by NMR spectroscopy. Our data represent the first structure determination of a C2 domain in its Ca2+-free and Ca2+-bound forms. Three Ca2+ ions were included in the Ca2+-bound structure, yielding a Ca2+-binding motif that involves five aspartate side chains and one serine side chain. Ca2+ immobilizes the structure of the C2A domain but does not produce a significant conformational change from a well-defined conformation to another. Thus, the mechanism of action of the C2A domain of synaptotagmin I is different from that used by Ca2+-binding proteins of the EF-hand family. The main effect of Ca2+ binding on the C2A domain is to change its electrostatic potential rather than its structure. These results support a model whereby the C2A domain functions as an electrostatic switch in neurotransmitter release. The similarity between the structures of the synaptotagmin I C2A domain and the PLC-delta1 C2 domain suggests that the latter binds four Ca2+ ions and acts by a similar mechanism. This mechanism may also be valid for other C2 domains that share the unusual ability to bind multiple Ca2+ ions in a tight cluster at the tip of the domain.
C2结构域是广泛存在的钙离子结合模块，在参与膜运输和信号转导的蛋白质中尤为丰富。突触结合蛋白I的C2A结构域被认为通过其与 syntaxin和磷脂的钙离子依赖性相互作用在神经递质释放中起关键作用。阐明钙离子与C2A结构域结合的结构后果对于理解其作用机制以及其他C2结构域的功能模型至关重要。我们通过核磁共振光谱法确定了突触结合蛋白I的C2A结构域的无钙和结合钙形式的溶液结构。我们的数据代表了首次对C2结构域的无钙和结合钙形式进行结构测定。结合钙的结构中包含三个钙离子，产生了一个涉及五个天冬氨酸侧链和一个丝氨酸侧链的钙离子结合基序。钙离子使C2A结构域的结构固定，但不会使其从一个明确的构象发生显著的构象变化。因此，突触结合蛋白I的C2A结构域的作用机制不同于EF手家族的钙离子结合蛋白所使用的机制。钙离子结合对C2A结构域的主要影响是改变其静电势而非其结构。这些结果支持了一个模型，即C2A结构域在神经递质释放中作为一个静电开关发挥作用。突触结合蛋白I的C2A结构域与PLC-δ1的C2结构域之间的结构相似性表明，后者结合四个钙离子并通过类似的机制起作用。这种机制可能对其他C2结构域也有效，这些结构域具有在结构域末端紧密簇中结合多个钙离子的特殊能力。
