虽然蛋白质构象异质性在生物学功能的许多方面都起着重要的作用。包括配体结合,其影响难以量化。大分子X射线衍射通常用静态结构来解释,但是它可以提供有关非调和和调和对构象异质性的贡献的信息。这里,通过时间和空间平均电子密度的多构象模型,我们测量了743对严格匹配的晶体学数据集的构象异质性,这些数据集反映了未结合/apo和配体结合/holo状态。当比较侧链的构象异质性时,我们观察到,当结合位点残基在配体结合时变得更加刚性时,远处的残留物往往会变得更加灵活,尤其是在非溶剂暴露区域。在配体性质中,我们观察到随着氢键数量的减少和相对疏水性的增加,蛋白质的柔韧性增加。在CDK2的一系列13种抑制剂结合结构中,我们发现构象异质性与抑制剂特征相关,并确定了构象变化如何在远离结合位点的构象异质性中传播差异。总的来说,我们的发现与核磁共振研究中出现的模型一致,这些模型表明残留侧链熵可以调节亲和力,并指出需要将静态构象变化和构象异质性整合到配体结合模型中。
蛋白质是我们细胞的主力。它们是“折叠”成特定的大分子,通常非常复杂,三维结构。这些结构不是静态的,而是动态和灵活的。换句话说,蛋白质可以在不同的三维形状之间转换,以在细胞内执行任务。为了发挥他们的作用,许多蛋白质必须与小分子配体结合。许多配体是药物,这意味着它们的有效性取决于它们结合并影响与所治疗疾病有关的蛋白质的能力。当配体与蛋白质结合时,它可以重塑蛋白质。例如,蛋白质的某些构象,蛋白质很难独立存在,当配体结合时可能变得更稳定。此外,在配体结合时,蛋白质的某些部分可以相对于彼此移动。先前的研究表明,这些运动可以影响配体和蛋白质之间的相互作用。然而,这些研究只检测了少量的蛋白质。因此,Wankowicz等人。着手确定,更详细地说,配体结合后蛋白质的灵活性会发生什么。首先,创建了一个管道来模拟有和没有配体连接的蛋白质的替代构型。这些模型测量了蛋白质结构内的灵活性。模型显示,当配体与蛋白质结合时,蛋白质不同区域的灵活性会发生变化-并且以一致的方式进行。在与配体相互作用的区域变得更刚性的蛋白质在其他区域变得不那么刚性,遥远的地区,反之亦然。换句话说,蛋白质的其余部分能够补偿配体结合引起的任何灵活性变化,这可能有助于配体与蛋白质结合的程度。这项研究证明了配体能够影响它们结合的蛋白质的整个结构,因此揭示了蛋白质的先天构象灵活性在这一过程中的作用。这些结果将有助于我们理解参与不同细胞过程的配体和蛋白质如何相互作用-并且,潜在的,如何操纵这些互动。
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 decreases 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 nuclear magnetic resonance 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.
Proteins are the workhorses of our cells. They are large molecules that ‘fold’ into specific, often highly complex, three-dimensional configurations. These structures are not static, but rather dynamic and flexible. In other words, proteins can shift between different three-dimensional shapes to perform their tasks within the cell. To perform their roles, many proteins have to bind to small molecule ligands. Many ligands are drugs, which means that their effectiveness depends on their ability to bind to and impact the proteins involved in the disease they are treating. When a ligand binds to a protein, it can reshape the protein. For example, certain conformations of the protein, which were difficult for the protein to be in on its own, may become more stable when the ligand binds. Additionally, upon ligand binding, some parts of the protein may move relative to each other. Previous studies have shown that these movements can affect the interaction between ligand and protein. However, these studies only examined a small number of proteins. Therefore, Wankowicz et al. set out to determine, in greater detail, what happens to protein flexibility upon ligand binding. First, a pipeline was created to model alternative configurations of the protein both with and without ligands attached. These models measured flexibility within protein structures. The models revealed that when ligands bind to proteins, the flexibility of different regions of the protein changes – and does so in a consistent way. Proteins that become more rigid in the region interacting with their ligands become less rigid in other, distant regions, and vice versa. In other words, the rest of the protein is able to compensate for any changes in flexibility caused by ligand binding, which may contribute to how well a ligand binds to a protein. This study demonstrates the ability of ligands to affect the entire structure of the proteins they bind to, and therefore sheds new light on the role of proteins’ innate conformational flexibility during this process. These results will contribute to our understanding of how the ligands and proteins involved in different cellular processes interact with each other – and, potentially, how these interactions can be manipulated.