Structural Flexibility of Cyclosporine A Is Mediated by Amide Cis–Trans Isomerization and the Chameleonic Roles of Calcium

Abstract

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 Ca2+, 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 CDCl3. To adopt these conformations, the macrocyclic ring has to twist and bend via cis–trans 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 CD3OD/H2O and 55% in CD3CN. However, upon interacting with Ca2+, in addition to the bent and previously reported closed conformations of free CycA, the CycA:Ca2+ complex is open and has all-trans peptide bonds. Previous NMR studies using calcium perchlorate reported only the closed conformation of CycA (which contains one cis peptide bond). Here, calcium chloride, a more biologically relevant salt, was used, and interestingly, it helps converting the cis -MeLeu9–MeLeu10 peptide bond into a trans 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 Ca2+:CycA complex may play an essential role in the binding of CycA to CypA.

Cyclosporin A: Conformational Complexity and Chameleonicity

“…Recently, Gray et al proposed the compact bent-twisted conformation of CsA via the results of the collision cross section (CCS) employing the distance geometry conformational sampling. 11 They confirmed that a minimum of two cis isomers (3−4 cis and 9−10 cis) existed in the conformation. This conformation corresponds to the position at (PC-1, PC-2) = (0.5, 0.45), where only AMBER12:EHT exhibited PMF ≈ 1.7 kcal/mol and the other force fields have PMF > 2.5 kcal/ mol using our bulk solvent simulations (Figure 2a and Figure S3), which is completely different from the one we found at (PC-1, PC-2) = (1.4, 0.0) that contains the same two cis isomers.…”

“…Expectedly, the canonical amino acids (Abu 2 , Val 5 , Ala 7 , and Dal 8 ) formed only trans isomers. Regarding the N-methyl amino acids, Mva 11 formed only a trans isomer, whereas the other N-methylated amino acids formed the cis and trans isomers.…”

“…2−11 The experimental results, as obtained by crystallography and NMR, reveal that CsA exhibits chameleonic behaviors, i.e., "closed," "open," "very open" conformations, etc., depending on the environment (solvent polarity, presence of metal ions or micelles, and complex formation with proteins). 2,3,12−18 A crystal structure in the closed conformation with four intramolecular hydrogen bonds (IMHBs; i.e., Abu 2 -H−Val 5 -O, Val 5 -H−Abu 2 -O, Ala 7 -H−Mva 11 -O, and Dal 8 -H−Mle 6 -O) has been reported (ID: DEKSAN, 2 source: CSD). A similarly closed conformation has also been reported in CDCl 3 .…”

“…21 A minimum of six sets of signals have been observed in methanol (MeOH). 22 Recently, Gray et al 11 proposed a bent conformation, which forms a more compact conformation than the canonical closed one via combined ionmobility spectrometry−mass spectrometry (IMS−MS), and proposed that it might exhibit multiple cis peptide bonds.…”

“…23 A fourth postulated binding mechanism of CsA to its partner protein(s) is a conformational change induced by metal ion binding. 11,24 Very recently, an excellent review of cyclosporin's structure and its permeability was published. 25 These authors summarized chemical structures, dynamics, and permeability data for cyclosporin A and its analogues from the literature and proposed two permeation models: (i) the simplified inducedfit-like (IFL) model and (ii) the conformational-selection-like (CSL) model, which is a modified version of the congruent model proposed by Witek et al; 8 membrane permeation is assisted by congruent conformations, which are significantly populated in water and the membrane.…”