Gastric proton pump with two occluded K(+) engineered with sodium pump-mimetic mutations.
Gastric proton pump with two occluded K(+) engineered with sodium pump-mimetic mutations.
复制标题
DOI：
10.1038/s41467-021-26024-1
复制
发表时间：
2021-09-29
影响因子：
16.6
通讯作者：
Oshima A
中科院分区：
文献类型：
--
作者：
Abe K;Yamamoto K;Irie K;Nishizawa T;Oshima A
胃 H+,K+-ATP 酶介导跨膜水解的 1H+/1K+ 的电中性交换。先前对 H+,K+-ATP 酶的 K+ 闭塞 E2-P 过渡态的结构分析表明，在阳离子结合位点 II 处有一个单一结合的 K+，与密切相关的 Na+,K+-ATP 酶的位点 I 和 II 处闭塞的两个 K+ 离子形成鲜明对比，后者介导跨膜电生 3Na+/2K+ 易位。这些 K+ 反向输送泵之间不同 K+ 化学计量的分子基础尚不清楚。基于钠泵的结构，我们展示了一系列在位点 I 附近发生变化的 H+,K+-ATPase 突变体的晶体结构和冷冻电镜结构。我们逐步定制的突变体构建最终得到了两个 K+ 结合的 H+,K+-ATP 酶，通过五个突变实现，包括直接协调 K+ 的氨基酸（Lys791Ser、Glu820Asp）、间接促进阳离子结合位点形成（Tyr340Asn、Glu936Val）和变构稳定 K+ 封闭构象（Tyr799Trp）。这种处于 K+ 封闭 E2-P 状态的五重突变体在其 2.6 Å 分辨率冷冻电镜结构中明确地在阳离子结合位点处显示出两种不同的密度。这些结果为了解两个密切相关的阳离子泵如何指定其阳离子结合位点上容纳的 K+ 数量提供了新的见解。胃 H+,K+-ATP 酶是一种质子泵，可创造胃腔的酸性环境，维持胃粘膜细胞膜上的高质子梯度。在这里，合理设计的 H+,K+-ATP 酶突变体的结构分析提供了对该和其他 P 型 ATP 酶阳离子结合化学计量和机制的深入了解。
The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site. The gastric H+,K+-ATPase is a proton pump that creates the acidic environment of the stomach lumen, maintaining high proton gradient across the gastric mucosa cell membrane. Here, structural analysis of rationally designed H+,K+-ATPase mutants provides insight into this and other P-type ATPases cation binding stoichiometry and mechanisms.
登录
查看更多内容
电子计数和束诱导运动校正可实现近原子分辨率的单粒子冷冻电镜。
影响因子：
48
作者：
Li, Xueming;Mooney, Paul;Zheng, Shawn;Booth, Christopher R.;Braunfeld, Michael B.;Gubbens, Sander;Agard, David A.;Cheng, Yifan
通讯作者：
Cheng, Yifan
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.5
作者：
Danko, S;Yamasaki, K;Toyoshima, C
通讯作者：
Toyoshima, C
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
14.8
作者：
通讯作者：
--