Price for Opening the Transient Specificity Pocket in Human Aldose Reductase upon Ligand Binding: Structural, Thermodynamic, Kinetic, and Computational Analysis

Insights into the thermodynamic and kinetic signature of the transient opening of a protein-binding pocket resulting from accommodation of suitable substituents attached to a given parent ligand scaffold are presented. As a target, we selected human aldose reductase, an enzyme involved in the development of late-stage diabetic complications. To recognize a large scope of substrate molecules, this reductase opens a transient specificity pocket. The pocket-opening step was studied by X-ray crystallography, microcalorimetry, and surface plasmon resonance using a narrow series of 2-carbamoyl-phenoxy-acetic acid derivatives. Molecular dynamics simulations suggest that pocket opening occurs only once an appropriate substituent is attached to the parent scaffold. Transient pocket opening of the uncomplexed protein is hardly recorded. Hydration-site analysis suggests that up to five water molecules entering the opened pocket cannot stabilize this state. Sole substitution with a benzyl group stabilizes the opened state, and the energetic barrier for opening is estimated to be ∼5 kJ/mol. Additional decoration of the pocket-opening benzyl substituent with a nitro group results in a huge enthalpy-driven potency increase; on the other hand, an isosteric carboxylic acid group reduces the potency 1000-fold, and binding occurs without pocket opening. We suggest a ligand induced-fit mechanism for the pocket-opening step, which, however, does not represent the rate-determining step in binding kinetics.

本研究阐明了蛋白质结合口袋（protein-binding pocket）因适配连接于既定母核配体骨架的合适取代基而发生瞬时开放的热力学与动力学特征。本研究选取人类醛糖还原酶（human aldose reductase）作为研究靶点，该酶参与糖尿病晚期并发症的发生发展过程。为识别多样化的底物分子，该还原酶可形成瞬时特异性结合口袋。本研究通过X射线晶体学、微量量热法及表面等离子体共振技术，针对一系列结构相近的2-氨甲酰基苯氧乙酸衍生物，对口袋开放过程展开了系统研究。分子动力学（Molecular dynamics）模拟结果显示，仅当母核骨架连接有合适取代基时，口袋开放才会发生；未结合配体的蛋白质几乎不会发生瞬时口袋开放。水合位点分析表明，进入开放口袋的至多5个水分子无法稳定该开放状态。仅通过苄基取代即可稳定口袋开放状态，经估算，口袋开放的能垒约为5 kJ/mol。若在该与口袋开放相关的苄基取代基上进一步引入硝基，可使活性产生显著的焓驱动提升；反之，若使用等排的羧酸基取代，则会使活性降低1000倍，且结合过程不会伴随口袋开放。我们提出，口袋开放过程遵循配体诱导契合机制，但该过程并非结合动力学中的限速步骤。