Kumar Anil, Manimekalai Malathy Sony Subramanian, Balakrishna Asha Manikkoth, Hunke Cornelia, Weigelt Sven, Sewald Norbert, Grüber Gerhard
Division of Structural and Computational Biology, Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
Proteins. 2009 Jun;75(4):807-19. doi: 10.1002/prot.22289.
A strategically placed tryptophan in position of Arg416 was used as an optical probe to monitor adenosine triphosphate and adenosine-diphosphate binding to subunit B of the A(1)A(O) adenosine triphosphate (ATP) synthase from Methanosarcina mazei Gö1. Tryptophan fluorescence and fluorescence correlation spectroscopy gave binding constants indicating a preferred binding of ATP over ADP to the protein. The X-ray crystal structure of the R416W mutant protein in the presence of ATP was solved to 2.1 A resolution, showing the substituted Trp-residue inside the predicted adenine-binding pocket. The cocrystallized ATP molecule could be trapped in a so-called transition nucleotide-binding state. The high resolution structure shows the phosphate residues of the ATP near the P-loop region (S150-E158) and its adenine ring forms pi-pi interaction with Phe149. This transition binding position of ATP could be confirmed by tryptophan emission spectra using the subunit B mutant F149W. The trapped ATP position, similar to the one of the binding region of the antibiotic efrapeptin in F(1)F(O) ATP synthases, is discussed in light of a transition nucleotide-binding state of ATP while on its way to the final binding pocket. Finally, the inhibitory effect of efrapeptin C in ATPase activity of a reconstituted A(3)B(3)- and A(3)B(R416W)(3)-subcomplex, composed of subunit A and the B subunit mutant R416W, of the A(1)A(O) ATP synthase is shown.
在嗜甲烷八叠球菌Gö1的A(1)A(O)三磷酸腺苷（ATP）合酶的亚基B中，一个处于精氨酸416位置的经过策略性定位的色氨酸被用作光学探针，以监测三磷酸腺苷（ATP）和二磷酸腺苷（ADP）与该亚基的结合情况。色氨酸荧光和荧光相关光谱给出的结合常数表明，ATP比ADP更倾向于与该蛋白质结合。在ATP存在的情况下，R416W突变蛋白的X射线晶体结构被解析到2.1埃的分辨率，显示出被取代的色氨酸残基位于预测的腺嘌呤结合口袋内。共结晶的ATP分子可以被困在所谓的过渡核苷酸结合状态。高分辨率结构显示，ATP的磷酸残基靠近P环区域（S150 - E158），其腺嘌呤环与苯丙氨酸149形成π-π相互作用。使用亚基B突变体F149W的色氨酸发射光谱可以证实ATP的这种过渡结合位置。根据ATP在进入最终结合口袋的过程中的过渡核苷酸结合状态，讨论了被困的ATP位置，该位置类似于F(1)F(O) ATP合酶中抗生素依弗帕肽结合区域的位置。最后，展示了依弗帕肽C对由A(1)A(O) ATP合酶的亚基A和亚基B突变体R416W组成的重组A(3)B(3)-和A(3)B(R416W)(3)-亚复合物的ATP酶活性的抑制作用。
