蛋白质在行使其生理功能时的无序化越来越受大家的关注。抗酸分子伴侣HdeA在行使其在酸性条件下保护蛋白质的功能时，其分子结构会变得无序。由于缺乏HdeA无序化的实验信息，HdeA无序化以及与其底物发生相互作用的机理仍不清楚。金长文教授课题组利用高场核磁共振在蛋白质动力学研究方面的优势，使用先进的NMR CEST实验，辅以其它常规NMR实验，表征出了HdeA在酸性条件下的激活状态，并找到与底物蛋白质相互作用的位点。该工作的主要实验结果均是在凤凰工程北大核磁平台的仪器上完成，彰显了高场核磁共振仪器在蛋白质动力学研究中具有的显著优势。并且其中部分CEST实验的实验方法也为国际首创。该项目是科技部国家重点研发项目《蛋白质机器瞬态构象的核磁共振研究》的一部分，阶段性成果已在《The Journal of Biological Chemistry》和《Journal of Magnet Resonace》等杂志上发表。
Structural basis and mechanism of the unfolding-induced activation of HdeA, a bacterial acid response chaperone
Yu XC, Hu Y, Ding J, Li H, Jin C.
Abstract
The role of proteinstructuraldisorder in biological functions has gained increasing attention in the past decade. Thebacterialacid-resistantchaperoneHdeAbelongs to a group of "conditionally disordered" proteins, because it is inactive in its well-structured state and becomes activated via an order-to-disorder transition underacidstress. However, themechanismforunfolding-inducedactivationremains unclear because of a lack of experimental information on the unfolded state conformation and thechaperone-client interactions. Herein, we used advanced solution NMR methods to characterize the activated-state conformation of HdeA under acidic conditions and identify its client-binding sites. We observed that the structure of activatedHdeAbecomes largely disordered and exposes two hydrophobic patches essential for client interactions. Furthermore, using the pH-dependent chemical exchange saturation transfer (CEST) NMR method, we identified threeacid-sensitive regions that act asstructurallocks in regulating the exposure of the two client-binding sites during theactivationprocess, revealing a multistepactivationmechanism of HdeA'schaperonefunction at the atomic level. Our results highlight the role of intrinsic protein disorder inchaperonefunction and the self-inhibitory role of ordered structures under nonstress conditions, offering new insights for improving our understanding of protein structure-function paradigms.
J Biol Chem.2019 Mar 1;294(9):3192-3206. doi: 10.1074/jbc.RA118.006398. Epub 2018 Dec 20
