Department of Biochemistry, Asahikawa Medical University, Midorigaoka-higashi 2-1-1-1, Asahikawa, 078-8510, Japan.
Sci Rep. 2022 Jul 16;12(1):12200. doi: 10.1038/s41598-022-16091-9.
Arg324 of sarcoplasmic reticulum Ca-ATPase forms electrostatic interactions with the phosphate moiety of phospholipids in most reaction states, and a hydrogen bond with Tyr122 in other states. Using site-directed mutagenesis, we explored the functional roles of Arg324 interactions, especially those with lipids, which at first glance might seem too weak to modulate the function of such a large membrane protein. The hydrogen bond forms transiently and facilitates Ca binding from the cytoplasmic side. The contributions of the electrostatic interactions to the reaction steps were quantified using a rate vs activity coefficient plot. We found that the interaction between Arg324 and lipids decreases the affinity for luminal Ca. The transformation rate of the phosphoenzyme intermediate is facilitated by the electrostatic interactions, and the function of these interactions depends not only on the type but also on the composition of the phospholipids. The properties observed in microsomes could not be reproduced with any single phospholipid, but with a mixture of phospholipids that mimics the native membrane. These results suggest the importance of swapping of the lipid partners of different headgroups in the reaction step. This study shows that Arg324 plays a role in the reaction cycle via complex intra-protein and protein-lipid interactions.
肌浆网 Ca-ATP 酶的 Arg324 与大多数反应状态下的磷脂的磷酸部分形成静电相互作用，并在其他状态下与 Tyr122 形成氢键。使用定点突变技术，我们探讨了 Arg324 相互作用的功能作用，特别是与脂质的相互作用，这些相互作用乍一看似乎太弱，无法调节如此大的膜蛋白的功能。氢键是瞬态形成的，有助于从细胞质侧结合 Ca。使用速率与活度系数图对静电相互作用在反应步骤中的贡献进行了量化。我们发现 Arg324 与脂质之间的相互作用降低了对腔钙的亲和力。磷酸酶中间体的转化速率通过静电相互作用得到促进，这些相互作用的功能不仅取决于磷脂的类型，还取决于其组成。在微粒体中观察到的性质不能用任何单一的磷脂来复制，但可以用模拟天然膜的磷脂混合物来复制。这些结果表明在反应步骤中不同头基的脂质伴侣交换的重要性。这项研究表明 Arg324 通过复杂的蛋白质内和蛋白质-脂质相互作用在反应循环中发挥作用。
