TRP TRNA LIGASE--X-RAY STUDIES OF THE CATALYTIC CYCLE

TRP TRNA 连接酶--催化循环的 X 射线研究

• 批准号: 6018930
• 负责人: Charles W. Carter
• 金额: $ 22.19万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-15 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018930
• 关键词: Bacillus stearothermophilus; SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; acylation; adenosine triphosphate; alanine; aminoacid tRNA ligase; aminoacyl tRNA; computer simulation; conformation; drug discovery /isolation; enzyme mechanism; enzyme structure; leucine; molecular dynamics; protein structure function; tryptophan; valine

Our goal is to correlate conformation changes during aminoacylation of tRNA by with specific recognition and substrate transformations. We wish to test the specific prediction that conformational changes observed for B stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) on proceeding from ligand-free enzyme to the Trp-5' AMP complex position the tRNA anticodon-binding site suitably for acyl-transfer, relative to the active site of the other monomer. To this end, we will solve new X-ray structures of TrpRS complexes with the cognate tRNA and with ATP. During the previous funding cycle we solved the ligand-free enzyme and complexes with tryptophan; an activated ground-state ternary complex with ATP and the species-specific inhibitor, indolmycin; the natural adenylate intermediate, Trp-5'AMP, and a product, tryptophanyl-2'3'-ATP. We will extend the resolution and experimental phases for the Trp-5' AMP complex to its diffraction limit which better than 1.7 Angstrom units, to precisely specific sidechain packing interactions between the N- terminal helix of the Rossmann-fold domain two domains of the monomer, which apparently couple active-site behavior to the distal anticodon binding site via Ile 16. Ile 16 will be mutated to valine, leucine, and alanine to test that hypothesis that this residue couples the small domain containing the anticodon-binding site to. We will finish refining each of the relevant structures from the above list, in order to produce a structural reaction profile for aminoacid activation and acylation of tRNA. Of special interest will be changes at the dimer interface introduced by binding the acceptor stem of the tRNA to one active site and the anticodon to a site on the other monomer. To explain how suppression with tryptophan occurs with an anticodon mutant of trRNA gin, we will compare the complex with the previously obtained for GlnRS. Prokaryote TrpRS is a potentially valuable target for anti-infective drug discovery, owing to the availability of a prokaryote-specific inhibitor, indolmycin. We will examine the structural bases for high- affinity binding by comparison of several such complexes, and the bases for specificity by extending our structural analysis to archebacterial and eukaryotic TrpRSs.

我们的目标是关联氨酰化过程中的构象变化 tRNA 通过特异性识别和底物转化。 我们 希望验证构象变化的具体预测 观察到嗜热脂肪芽胞杆菌色氨酸-tRNA 合成酶 (TrpRS) 从无配体酶到 Trp-5' AMP 复合物位置 适合酰基转移的 tRNA 反密码子结合位点，相对于 另一个单体的活性位点。 为此，我们将解决新的 TrpRS 与同源 tRNA 和 ATP 复合物的 X 射线结构。 在上一个融资周期中，我们解决了无配体酶和 与色氨酸的复合物；激活的基态三元配合物 含有 ATP 和物种特异性抑制剂吲哚霉素；自然的 腺苷酸中间体 Trp-5'AMP 和产物色氨酸-2'3'-ATP。 我们将延长 Trp-5' AMP 的分辨率和实验阶段 复杂到其衍射极限，优于 1.7 埃单位， N-之间精确特定的侧链包装相互作用 罗斯曼折叠结构域的末端螺旋是单体的两个结构域， 显然将活性位点行为与远端反密码子结合起来 通过 Ile 16 的结合位点。Ile 16 将突变为缬氨酸、亮氨酸和 丙氨酸来检验该残基与小分子偶联的假设 包含反密码子结合位点的结构域。 我们将完成 细化上面列表中的每个相关结构，以便 产生氨基酸活化的结构反应谱 tRNA 的酰化。 特别令人感兴趣的是二聚体的变化 通过将 tRNA 的受体茎与一个受体结合而引入的界面 活性位点和另一个单体上位点的反密码子。 到 解释反密码子突变体如何发生色氨酸抑制 trRNA gin，我们将复合物与之前获得的进行比较 对于 GlnRS。 原核生物 TrpRS 是一个潜在有价值的抗感染靶点 药物发现，由于原核生物特异性的可用性 抑制剂，吲哚霉素。 我们将研究高水平的结构基础 通过比较几个此类复合物和碱基来进行亲和结合 通过将我们的结构分析扩展到古细菌来获得特异性 和真核 TrpRS。