Structure-Affinity Properties of a High-Affinity Ligand of FKBP12 Studied by Molecular Simulations of a Binding Intermediate

With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant of this intermediate should be decreased, resulting in an increase of the affinity constant . The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i) the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii) symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state.

为阐明FK506结合蛋白12（FKBP12）配体（ligand）的结构-亲和力特性，我们表征了该蛋白与高亲和力配体之间的结合中间态（binding intermediate state）。事实上，此类物种中形成的分子间接触（intermolecular contacts）的性质与范围，可能会影响其稳定性，进而影响整体结合速率。为找到该结合中间态，我们采用了一套分子模拟（molecular simulation）方案，通过逐步降低引入的偏置力（biasing forces）来实现配体的解离过程。随后，我们通过17次独立的随机边界分子动力学（stochastic boundary molecular dynamics）模拟对该中间态进行精修，最终得到了该中间态的一致结构图景。在该中间态中，配体的核心区域保持稳定，这主要得益于两个锚定氧原子——这类原子是所有FKBP12配体核心结构中均存在的保守基序（recurrent motifs）。此外，配体的非核心区域参与了大量瞬时性的分子间与分子内接触。多数接触的动态特性，对于配体保留至少部分构象熵（configurational entropy），以及避免结合路径上出现捕获态均至关重要。由于瞬时接触与锚定接触有助于提高中间态的稳定性，因此该中间态的解离速率常数（dissociation rate constant）应会降低，进而导致亲和力常数（affinity constant）升高。本研究结果印证了我们此前的结论，并为解释高亲和力配体的两大特征提供了连贯的理论依据：一是存在于不同核心结构的羰基（carbonyl group）或磺酰基（sulfonyl group）中的两个氧原子，二是作为非核心部分存在的对称或拟对称（pseudo-symmetric）可移动原子基团。该中间态的另一有趣特征是，该蛋白的柔性80s环（主要是其顶端区域）发生构象形变，这能够促进配体抵达结合态的可及性。