Ligand binding remodels protein side chain conformational heterogeneity

While protein conformational heterogeneity plays an important role in many aspects of biological function, including ligand binding, its impact has been difficult to quantify. Macromolecular X-ray diffraction is commonly interpreted with a static structure, but it can provide information on both the anharmonic and harmonic contributions to conformational heterogeneity. Here, through multiconformer modeling of time- and space-averaged electron density, we measure conformational heterogeneity of 743 stringently matched pairs of crystallographic datasets that reflect unbound/apo and ligand-bound/holo states. When comparing the conformational heterogeneity of side chains, we observe that when binding site residues become more rigid upon ligand binding, distant residues tend to become more flexible, especially in non-solvent exposed regions. Among ligand properties, we observe increased protein flexibility as the number of hydrogen bonds decrease and relative hydrophobicity increases. Across a series of 13 inhibitor bound structures of CDK2, we find that conformational heterogeneity is correlated with inhibitor features and identify how conformational changes propagate differences in conformational heterogeneity away from the binding site. Collectively, our findings agree with models emerging from NMR studies suggesting that residual side chain entropy can modulate affinity and point to the need to integrate both static conformational changes and conformational heterogeneity in models of ligand binding.

尽管蛋白质构象异质性（conformational heterogeneity）在包括配体结合（ligand binding）在内的诸多生物学功能环节中发挥着关键作用，但其影响却始终难以量化。常规上，大分子X射线衍射（Macromolecular X-ray diffraction）分析基于静态结构进行解析，但该技术可提供构象异质性的非简谐与简谐贡献相关信息。本研究通过对时间与空间平均电子密度（time- and space-averaged electron density）开展多构象建模（multiconformer modeling），对743组严格匹配的晶体衍射数据集（crystallographic datasets）对的构象异质性进行定量分析，这些数据集对分别对应蛋白质的未结合/脱辅基（apo）态与配体结合/全辅基（holo）态。在对比侧链（side chains）构象异质性时，我们观察到：当配体结合使结合位点残基（binding site residues）的刚性提升时，远端残基往往会变得更柔性，这一现象在非溶剂暴露区域（non-solvent exposed regions）尤为显著。在配体属性层面，我们发现随着氢键（hydrogen bonds）数量减少、相对疏水性（relative hydrophobicity）升高，蛋白质的柔性会随之增强。针对13组CDK2抑制剂结合结构的分析显示，构象异质性与抑制剂特征存在相关性；本研究还阐明了构象变化如何将构象异质性的差异从结合位点向外传递。综上，本研究的发现与核磁共振（NMR）领域新兴模型的结论一致——这些模型提出残余侧链熵可调控结合亲和力；同时本研究也指出，在配体结合模型中需同时整合静态构象变化与构象异质性两类因素。