Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, United States.
Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama, Japan.
Elife. 2019 May 3;8:e44760. doi: 10.7554/eLife.44760.
Ca-stimulated translocation of cytosolic phospholipase Aα (cPLAα) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLAα preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLAα C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl--glycero-3-phosphocholine (DHPC) and Ca ions. Two Ca are complexed at previously reported locations in the lipid-free C2-domain. One of these Caions, along with a third Ca, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation-π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLAα activity. The DHPC-binding mode of the cPLAα C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains.
钙刺激胞质型磷脂酶 Aα（cPLAα）向高尔基体易位诱导花生四烯酸产生，这是炎症性类二十烷酸合成的限速步骤。cPLAα 对富含磷脂酰胆碱（PC）的膜的偏好结构见解仍然难以捉摸。在这里，我们报告了 cPLAα C2 结构域（分辨率为 2.2Å）的结构，其中包含结合的 1,2-二己酰基 - 甘油-3-磷酸胆碱（DHPC）和 Ca 离子。两个 Ca 离子结合在脂质自由 C2 结构域中先前报道的位置。这些 Ca 离子之一，以及第三个 Ca 离子，将 C2 结构域桥接到 DHPC 磷酸基团，该基团还与 Asn65 相互作用。Tyr96 通过与 PC 三甲铵基团的阳离子-π 相互作用，在通过 C2 结构域识别脂质头部基团方面发挥关键作用。突变分析证实 Tyr96 和 Asn65 在 C2 结构域的 PC 结合选择性和 cPLAα 活性的调节中发挥作用。cPLAα C2 结构域与其他 C2 结构域结合的磷脂酰丝氨酸或磷脂酰肌醇 4,5-二磷酸不同，DHPC 的结合模式扩展并加深了对 C2 结构域介导的脂质结合机制的认识。
