Department of Chemistry, University of Colorado Denver, Denver, Colorado.
Department of Chemistry, University of Colorado Denver, Denver, Colorado.
Biophys J. 2019 Mar 19;116(6):1025-1036. doi: 10.1016/j.bpj.2019.01.035. Epub 2019 Feb 5.
Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP) with greater Ca sensitivity than either of its individual domains and binds to membranes containing PIP even in the absence of Ca. Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.
突触融合蛋白-1（Syt-1）和突触融合蛋白-7（Syt-7）含有相似的串联 C2 结构域，C2A 和 C2B，它们共同感应 Ca 来结合膜并促进胞吐融合孔的稳定。Syt-1 触发神经递质的快速释放，而 Syt-7 在涉及较低 Ca 浓度的过程中发挥作用，如激素分泌。据报道，Syt-1 的 C2 结构域以协同方式结合膜，这是基于它们作为 C2AB 串联比作为单个 C2 结构域插入膜中更深的观察结果。相比之下，我们之前基于对其脂质体解离动力学的测量结果，提出 Syt-7 的两个 C2 结构域独立结合膜的观点。在这里，我们使用可直接比较的测量方法研究了 Syt-1 和 Syt-7 的 C2A-C2B 结构域间的协同作用。平衡 Ca 滴定表明，Syt-7 的 C2AB 串联在缺乏磷脂酰肌醇-4,5-二磷酸（PIP）的脂质体上结合具有比其单个结构域更高的 Ca 敏感性，并且即使在没有 Ca 的情况下也可以结合含有 PIP 的膜。停流动力学测量显示 Syt-1 和 Syt-7 之间的协同作用存在差异：Syt-1 C2AB 从无 PIP 的脂质体中解离的速度比其单个 C2 结构域慢得多，表明存在协同作用，而 Syt-7 C2AB 的主要群体具有与 C2A 结构域相当的解离速率，表明缺乏协同作用。Syt-7 C2AB 的一小部分亚群体以较慢的速率解离，这可能是由于存在较小的协同成分和/或脂质体聚集。使用环境敏感荧光探针的测量表明，Syt-7 的 C2B 结构域作为 C2AB 串联的一部分深入插入膜中，类似于先前报道的 Syt-1 的共插入。总体而言，与 Syt-7 相比，Syt-1 的 C2A 和 C2B 结构域的共插入与协同能量效应的耦合程度要大得多。这种差异可以根据两个蛋白质中 C2A 和 C2B 结构域对膜结合的相对贡献来理解。
