ADP bound to K46bE mutant ATP-grasp fold of Blastocystis hominis succinyl-CoA synthetase

IF 2.2 4区 生物学
J. Huang, V. H. Nguyen, K. A. Hamblin, R. Maytum, M. V. D. Glezen, M. Fraser
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引用次数: 0

Abstract

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46βE, K114βD, V113βL and L227βF, and one with two mutations, K46βE/K114βD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20β, Gly111β and Val113β. The O atom of the side chain of Gln20β interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111β. It is the different conformations of the backbone at Gln20β, of the side chain of Gln20β and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227βF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46βE/K114βD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.
ADP与人胚囊虫琥珀酰辅酶a合成酶K46bE突变体atp把握褶结合
琥珀酰辅酶a合成酶(SCS)催化三羧酸循环中导致底物水平磷酸化的唯一步骤。某些形式的SCS对ADP/ATP或GDP/GTP具有特异性,而其他形式的SCS可以结合所有这些核苷酸,通常具有不同的亲和力。“守门人”残基理论已被提出来解释核苷酸特异性。看门人残基位于结合位点之外,与进入的核苷酸产生特定的静电相互作用,以确定核苷酸是否可以进入结合位点。为了验证这一理论,我们测定了Mg2+-ADP复合物中核苷酸结合域的晶体结构,以及K46βE、K114βD、V113βL和L227βF四个单突变蛋白的结构,以及K46βE/K114βD两个突变蛋白的结构。晶体结构表明,由于核苷酸与结合位点之间的相互作用,该酶对ADP/ATP具有特异性。核苷酸特异性是由腺嘌呤碱基与Gln20β、Gly111β和Val113β之间的氢键相互作用提供的。Gln20β侧链的O原子与ADP的N6相互作用,而侧链的N原子与Gly111β的羰基O原子相互作用。Gln20β的主链、Gln20β的侧链和连接体的不同构象使酶具有atp特异性。该连接子连接atp抓握折叠的两个亚域,并与腺嘌呤和鸟嘌呤碱基进行不同的相互作用。突变蛋白具有相似的构象,尽管L227βF突变体显示出破坏镁离子结合位点的结构变化。虽然根据动力学测定,人胚囊虫SCS的K46βE/K114βD双突变体对GTP的结合优于ATP,但只获得了与Mg2+-ADP的复合物。
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来源期刊
自引率
13.60%
发文量
0
审稿时长
3 months
期刊介绍: Acta Crystallographica Section D welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules or the methods used to determine them. Reports on new structures of biological importance may address the smallest macromolecules to the largest complex molecular machines. These structures may have been determined using any structural biology technique including crystallography, NMR, cryoEM and/or other techniques. The key criterion is that such articles must present significant new insights into biological, chemical or medical sciences. The inclusion of complementary data that support the conclusions drawn from the structural studies (such as binding studies, mass spectrometry, enzyme assays, or analysis of mutants or other modified forms of biological macromolecule) is encouraged. Methods articles may include new approaches to any aspect of biological structure determination or structure analysis but will only be accepted where they focus on new methods that are demonstrated to be of general applicability and importance to structural biology. Articles describing particularly difficult problems in structural biology are also welcomed, if the analysis would provide useful insights to others facing similar problems.
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