Gray Amber L H, Steren Carlos A, Haynes Isaac W, Bermejo Guillermo A, Favretto Filippo, Zweckstetter Markus, Do Thanh D
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States.
Computational Biomolecular Magnetic Resonance Core, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
J Phys Chem B. 2021 Feb 11;125(5):1378-1391. doi: 10.1021/acs.jpcb.0c11152. Epub 2021 Feb 1.
Falling outside of Lipinski's rule of five, macrocyclic drugs have accessed unique binding sites of their target receptors unreachable by traditional small molecules. Cyclosporin(e) A (CycA), an extensively studied macrocyclic natural product, is an immunosuppressant with undesirable side effects such as electrolytic imbalances. In this work, a comprehensive view on the conformational landscape of CycA, its interactions with Ca, and host-guest interactions with cyclophilin A (CypA) is reported through exhaustive analyses that combine ion-mobility spectrometry-mass spectrometry (IMS-MS), nuclear magnetic resonance (NMR) spectroscopy, distance-geometry modeling, and NMR-driven molecular dynamics. Our IMS-MS data show that CycA can adopt extremely compact conformations with significantly smaller collisional cross sections than the closed conformation observed in CDCl. To adopt these conformations, the macrocyclic ring has to twist and bend via - isomerization of backbone amides, and thus, we termed this family of structures the "bent" conformation. Furthermore, NMR measurements indicate that the closed conformation exists at 19% in CDOD/HO and 55% in CDCN. However, upon interacting with Ca, in addition to the bent and previously reported closed conformations of free CycA, the CycA:Ca complex is open and has all- peptide bonds. Previous NMR studies using calcium perchlorate reported only the closed conformation of CycA (which contains one peptide bond). Here, calcium chloride, a more biologically relevant salt, was used, and interestingly, it helps converting the -MeLeu9-MeLeu10 peptide bond into a bond. Last, we were able to capture the native binding of CycA and CypA to give forth evidence that IMS-MS is able to probe the solution-phase structures of the complexes and that the Ca:CycA complex may play an essential role in the binding of CycA to CypA.
大环药物不符合利平斯基五规则，能够作用于传统小分子无法触及的靶受体独特结合位点。环孢菌素A（CycA）是一种经过广泛研究的大环天然产物，是一种免疫抑制剂，具有诸如电解质失衡等不良副作用。在这项工作中，通过结合离子淌度质谱（IMS-MS）、核磁共振（NMR）光谱、距离几何建模和NMR驱动的分子动力学的详尽分析，报道了关于CycA构象景观、其与Ca的相互作用以及与亲环蛋白A（CypA）的主客体相互作用的全面观点。我们的IMS-MS数据表明，CycA可以采用极其紧凑的构象，其碰撞横截面明显小于在CDCl中观察到的封闭构象。为了采用这些构象，大环必须通过主链酰胺的-异构化发生扭曲和弯曲，因此，我们将这个结构家族称为“弯曲”构象。此外，NMR测量表明，封闭构象在CDOD/HO中占19%，在CDCN中占55%。然而，与Ca相互作用时，除了游离CycA的弯曲构象和先前报道的封闭构象外，CycA:Ca复合物是开放的且所有肽键均为。先前使用高氯酸钙的NMR研究仅报道了CycA的封闭构象（其包含一个肽键）。在这里，使用了氯化钙，一种更具生物学相关性的盐，有趣的是，它有助于将-MeLeu9-MeLeu10肽键转化为键。最后，我们能够捕捉到CycA和CypA的天然结合，从而证明IMS-MS能够探测复合物的溶液相结构，并且Ca:CycA复合物可能在CycA与CypA的结合中起重要作用。
