IDIBE, Universidad Miguel Hernández, Elche, Alicante, Spain; Instituto de Biocomputación y Física de Sistemas Complejos (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, Zaragoza, Spain.
Department of Life and Environmental Sciences, Marche Polytechnic University, Ancona, Italy.
Arch Biochem Biophys. 2020 Apr 15;683:108303. doi: 10.1016/j.abb.2020.108303. Epub 2020 Feb 16.
Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R, R and η), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules.
谷氨酰胺合成酶（GS）催化 ATP 依赖性地将谷氨酸和氨转化为谷氨酰胺。Synechocystis sp. PCC 6803 GS 的活性受到多种机制的调节，包括与一种 65 个残基长的、固有无序蛋白（IDP）IF7 的蛋白-蛋白相互作用。IDP 在其自由状态和在某些情况下在其分子复合物中探索多种构象。我们使用 11.7 T 核磁共振（NMR）和小角度 X 射线散射（SAXS）来研究：（i）分离的 IF7；（ii）IF7/GS 复合物的大小和皮秒到纳秒（ps-ns）时间尺度的动力学。我们的 SAXS 发现，与 MD 结果一起，表明：（i）溶液中 IF7 的一些可能结构；（ii）IF7 的存在影响了 GS 在溶液中的结构。SAXS 和 NMR 的联合使用表明，IF7 的每个氨基酸的运动与其相邻氨基酸的运动无关。在游离和结合物种中，IF7 的 R、R 和 η 值最大的残基主要聚集在：（i）该蛋白的 C 末端；和（ii）Ala30。这些残基与 Arg8（与 GS 相互作用的热点残基）一起，在存在 GS 时具有受限的流动性。在我们对分离的 IF7 的 MD 模拟中，C 末端区域似乎更紧凑，似乎参与与 GS 的非天然接触，这有助于两个大分子之间的结合。
