BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR

Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach.

蛋白质-蛋白质相互作用是一类兼具挑战性与愈发重要性的治疗性化合物设计靶点，此类化合物可用于干预生物体内的生理过程。近年来，基于片段的策略被提出，作为从类片段分子开发蛋白质-蛋白质界面抑制剂的极具吸引力的途径。靶向蛋白质-蛋白质相互作用的一大核心挑战，与分子识别过程中蛋白质表面的结构适应性变化密切相关。因此，在药物研发的早期阶段，亟需能够识别片段结合后蛋白质细微构象变化的方法。本研究报道了一种快速核磁共振（Nuclear Magnetic Resonance, NMR）方法，可用于探测片段结合后蛋白质发生的细微构象变化。该方法基于将实验测得的片段诱导的胺基质子化学位移扰动（Chemical Shift Perturbation, CSP），与针对一系列对接片段构象所模拟得到的CSP进行比对，同时考量片段结合产生的环电流效应。我们通过对抗凋亡蛋白Bcl-xL与片段4'-氟-[1,1'-联苯]-4-羧酸的复合物进行回顾性分析，对该方法进行了验证；此前已有研究表明，该片段可结合至Bcl-xL的一个热点结合区域。基于CSP的分析结果表明，该蛋白质在与片段结合后，其位于第2、3螺旋以及第5螺旋起始区域的残基发生了细微的构象重排。我们的观测结果通过对游离态与片段结合态Bcl-xL蛋白进行的残余偶极耦合（Residual Dipolar Coupling）测量得到了验证。这些基于NMR的实验结果，与此前的分子动力学计算结果完全一致；后者证实了Bcl-xL在结合位点附近具有较高的柔性。本研究证实，蛋白质胺基质子的CSP是一类实用且可靠的结构探测探针。因此，我们提出在基于片段的药物研发策略中，可通过CSP模拟来评估配体结合后蛋白质的构象变化。