Dmitriev Oleg Y, Abildgaard Frits, Markley John L, Fillingame Robert H
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Biochemistry. 2002 Apr 30;41(17):5537-47. doi: 10.1021/bi012198l.
The structure of the A24D/D61N substituted subunit c of Escherichia coli ATP synthase, in which the essential carboxylate has been switched from residue 61 of the second transmembrane helix (TMH) to residue 24 of the first TMH, has been determined by heteronuclear multidimensional NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture. As in the case of the wild-type protein, A24D/D61N substituted subunit c forms a hairpin of two extended alpha-helices (residues 5-39 and 46-78), with residues 40-45 forming a connecting loop at the center of the protein. The structure was determined at pH 5, where Asp24 is fully protonated. The relative orientation of the two extended helices in the A24D/D61N structure is different from that in the protonated form of the wild-type protein, also determined at pH 5. The C-terminal helix is rotated by 150 degrees relative to the wild-type structure, and the N-terminal helix is rotated such that the essential Asp24 carboxyl group packs on the same side of the molecule as Asp61 in the wild-type protein. The changes in helix-helix orientation lead to a structure that is quite similar to that of the deprotonated form of wild-type subunit c, determined at pH 8. When a decameric ring of c subunits was modeled from the new structure, the Asp24 carboxyl group was found to pack in a cavity at the interface between two subunits that is similar to the cavity in which Asp61 of the wild-type protein is predicted to pack. The interacting faces of the packed subunits in this model are also similar to those in the wild-type model. The results provide further evidence that subunit c is likely to fold in at least two conformational states differing most notably in the orientation of the C-terminal helix. Based upon the structure, a mechanistic model is discussed that indicates how the wild-type and A24D/D61N subunits could utilize similar helical movements during H(+) transport-coupled rotation of the decameric c ring.
已通过异核多维核磁共振在单相氯仿/甲醇/水（4:4:1）溶剂混合物中确定了大肠杆菌ATP合酶A24D/D61N替代亚基c的结构，其中关键羧酸盐已从第二个跨膜螺旋（TMH）的61位残基切换到第一个TMH的24位残基。与野生型蛋白质的情况一样，A24D/D61N替代亚基c形成了由两个延伸的α螺旋（残基5 - 39和46 - 78）组成的发夹结构，残基40 - 45在蛋白质中心形成连接环。该结构是在pH 5时确定的，此时Asp24完全质子化。A24D/D61N结构中两个延伸螺旋的相对取向与同样在pH 5时确定的野生型蛋白质质子化形式中的不同。C末端螺旋相对于野生型结构旋转了150度，N末端螺旋旋转使得关键的Asp24羧基与野生型蛋白质中的Asp61在分子的同一侧堆积。螺旋 - 螺旋取向的变化导致了一种与在pH 8时确定的野生型亚基c去质子化形式的结构非常相似的结构。当根据新结构构建c亚基的十聚体环模型时，发现Asp24羧基堆积在两个亚基之间界面处的一个腔中，该腔类似于预测野生型蛋白质的Asp61会堆积的腔。此模型中堆积亚基的相互作用面也与野生型模型中的相似。结果提供了进一步的证据，表明亚基c可能以至少两种构象状态折叠，最显著的区别在于C末端螺旋的取向。基于该结构，讨论了一个机制模型，该模型表明野生型和A24D/D61N亚基在十聚体c环的H(+)转运偶联旋转过程中如何利用相似的螺旋运动。
