Department of Biochemistry and Biophysics, Oregon State University , Corvallis, Oregon 973331, United States.
Biochemistry. 2014 Aug 5;53(30):5023-33. doi: 10.1021/bi5004469. Epub 2014 Jul 23.
Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25-95 μM) and low affinities (Kd = 400-700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'-myoinositol-4',5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.
外排蛋白是一种由六个 C2 结构域组成的跨膜蛋白，被认为是胞吐作用的钙传感器。尽管外排蛋白被认为结合钙和脂质，但脂质的特异性和钙结合结构域的身份存在争议。此外，目前尚不清楚外排蛋白的钙结合亲和力是否与已知引发胞吐作用的 30-50 μM 的最大细胞内突触前钙浓度相匹配。为了表征外排蛋白的钙和脂质结合特性，我们使用了等温滴定量热法（ITC）、脂质体沉淀测定法和荧光光谱法。ITC 数据分析表明，除 C2A 结构域外，C2 结构域在溶液中以中等亲和力（Kd=25-95 μM）和低亲和力（Kd=400-700 μM）结合多个钙离子。然而，在脂质体存在的情况下，结构域的钙敏感性增加了 10 倍。还确定钙增强了结构域 C2B-C2E 与脂质体的结合，而 C2F 结构域以钙独立的方式与脂质体结合。破坏 C2F 结构域钙结合的突变不会破坏脂质体结合，这支持了 C2F 结构域与磷脂酰丝氨酸相互作用的钙独立性结论。此外，结构域 C2C 和 C2F，而不是结构域 C2A、C2B、C2D 和 C2E，与二油酰基-sn-甘油-3-磷酸（1'-肌醇-4'，5'-二磷酸）[PI（4，5）P2]结合，这优先将它们导向含有 PI（4，5）P2 的脂质体。值得注意的是，C2C 中的赖氨酸突变 L478A 和 L480A 选择性地削弱了 PI（4，5）P2 的相互作用，而不影响磷脂酰丝氨酸的结合。最后，环境敏感荧光非天然氨基酸发射光谱的位移表明，C2F 结构域的钙结合环以钙独立的方式直接与带负电荷的脂质体的脂质双层相互作用。基于这些结果，我们提出外排蛋白的 C2F 和 C2C 结构域优先结合 PI（4，5）P2，并且 PI（4，5）P2 可能以钙独立的方式将外排蛋白靶向突触前。这种定位将促进毛细胞突触的快速钙依赖性胞吐作用。
