Chemical conformation of the essential glutamate site of the c-ring within thermophilic bacillus FoF1-ATP synthase determined by solid-state NMR based on its isolated c-ring structure.
Chemical conformation of the essential glutamate site of the c-ring within thermophilic bacillus FoF1-ATP synthase determined by solid-state NMR based on its isolated c-ring structure.
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根据分离的 C 环结构，通过固态 NMR 确定嗜热芽孢杆菌 FoF1-ATP 合酶内 C 环必需谷氨酸位点的化学构象。
DOI：
10.1021/jacs.2c03580
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发表时间：
2022
影响因子：
15
通讯作者：
H.
中科院分区：
文献类型：
--
作者：
Todokoro;Y.;Kang;S.J.;Suzuki;T.;Ikegami;T.;Kainosho;M.;Yoshida;M.;Fujiwara;T.;Akutsu;H.
通过F型ATP合酶（FoF 1）的膜包埋Fo组分的质子转运由Fo亚基环（c环）的旋转促进，其在必需酸性氨基酸残基处携带质子。FoF 1的冷冻电子显微镜（Cryo-EM）结构表明了独特的质子转运机制。为了阐明它的基础上的FoF 1的环的必需的酸性残基的化学构象，我们确定了分离的thermophilicBacillusFo（tFo）的C-环的结构，由10个亚基，在膜中的固态NMR。该结构包含独特的质子锁定构象，其中Asn 23（cN23）CγO和Glu 56（cE 56）CδOH以闭合形式形成氢键。我们将立体阵列同位素标记的Glu和Asn引入tFoc环中，以阐明tFoF 1-ATP合酶（tFoF 1）中这些残基的化学构象。在分子量为505 kDa的膜蛋白复合物中，cN23和cE 56分别检测到两个分离良好的~（13）C信号，表明存在两种不同的化学构象。根据tFoc-ring和tFoF 1的信号强度和结构，被膜脂包围的cN23和cE 56中有6对呈封闭形式，而在a-c界面的另外4对呈去质子化的开放形式，占87%。这表明a-c界面是高度亲水的。根据N23在开放和封闭形式下的信号强度以及每个cE 56周围极性残基的分布来估计a-c界面中四个cE 56残基的pKa值。这一结果有利于ATP的合成。
Proton translocation through the membrane-embedded Focomponent of F-type ATP synthase (FoF1) is facilitated by the rotation of the Foc-subunit ring (c-ring), carrying protons at essential acidic amino acid residues. Cryo-electron microscopy (Cryo-EM) structures of FoF1suggest a unique proton translocation mechanism. To elucidate it based on the chemical conformation of the essential acidic residues of thec-ring in FoF1, we determined the structure of the isolated thermophilicBacillusFo(tFo)c-ring, consisting of 10 subunits, in membranes by solid-state NMR. This structure contains a distinct proton-locking conformation, wherein Asn23 (cN23) CγO and Glu56 (cE56) CδOH form a hydrogen bond in a closed form. We introduced stereo-array-isotope-labeled (SAIL) Glu and Asn into the tFoc-ring to clarify the chemical conformation of these residues in tFoF1-ATP synthase (tFoF1). Two well-separated13C signals could be detected forcN23 andcE56 in a 505 kDa membrane protein complex, respectively, thereby suggesting the presence of two distinct chemical conformations. Based on the signal intensity and structure of the tFoc-ring and tFoF1, six pairs ofcN23 andcE56 surrounded by membrane lipids take the closed form, whereas the other four in thea–cinterface employ the deprotonated open form at a proportion of 87%. This indicates that thea–cinterface is highly hydrophilic. The pKavalues of the fourcE56 residues in thea–cinterface were estimated from thecN23 signal intensity in the open and closed forms and distribution of polar residues around eachcE56. The results favor a rotation of thec-ring for ATP synthesis.