Department of Chemistry, University of Saskatchewan, Saskatoon, SK S7N5C9, Canada.
Protein Sci. 2011 Jun;20(6):1021-9. doi: 10.1002/pro.635. Epub 2011 May 3.
In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR-Trx interface suggested that only four residues (F81, R130, F141, and F142) account for the majority of the EC TrxR-Trx interface stability. Individual replacement of equivalent residues in DR TrxR (M84, K137, F148, and F149) with alanine resulted in drastic changes in binding affinity, confirming that the four residues account for most of TrxR-Trx interface stability. When M84 and K137 were changed to match equivalent EC TrxR residues (K137R and M84F), the DR TrxR substrate specificity was altered from its own Trx to that of EC Trx. The results suggest that a small subset of the TrxR-Trx interface residues is responsible for the majority of Trx binding affinity and species-specific recognition.
在这项研究中，我们以大肠杆菌硫氧还蛋白还原酶（EC TrxR）与其底物硫氧还蛋白（Trx）复合物的 X 射线晶体结构为指导，设计了一种具有改变的 Trx 特异性的抗辐射球菌硫氧还蛋白还原酶（DR TrxR）突变体。先前的研究表明，TrxRs 对同源 Trxs（同种）的亲和力高于对不同种属 Trxs 的亲和力。计算丙氨酸扫描突变和 EC TrxR-Trx 界面的直观检查表明，只有四个残基（F81、R130、F141 和 F142）决定了 EC TrxR-Trx 界面的大部分稳定性。在 DR TrxR 中单独用丙氨酸替换等效残基（M84、K137、F148 和 F149）会导致结合亲和力发生剧烈变化，这证实了这四个残基决定了 TrxR-Trx 界面的大部分稳定性。当 M84 和 K137 被改变为与 EC TrxR 的等效残基（K137R 和 M84F）匹配时，DR TrxR 的底物特异性从自身 Trx 改变为 EC Trx。结果表明，TrxR-Trx 界面残基的一小部分决定了 Trx 的大部分结合亲和力和种属特异性识别。
