An atomic view including hydrogen positions of a main aqueous conformation of cyclosporine A (CycA), an important 11-amino acid macrocyclic immunosuppressant, is reported for the first time. Until now, the “closed” form of CycA in non-polar solvent and the “open” form in the CycA:cyclophilin A (CycA:CypA) complex were known to exist in solution. For decades, it has been a grand challenge to determine the conformation of free CycA in aqueous solution given its poor solubility in water. Using a combination of X-ray crystallography and single-crystal neutron diffraction, we unambiguously resolve a unique conformer with a novel cis-amide between residues 11 and 1 of CycA and two water ligands that stabilize both intra- and intermolecular hydrogen bond networks. Nuclear magnetic resonance (NMR) and titration experiments indicate that the novel conformer exists in solution as a main conformer. It is as abundant as the closed conformer in 90/10 methanol/water and is the main conformer at 10/90 methanol/water. There is no evidence supporting the open form of unbound CycA with all trans amides existing in water. The conformation with cis MeVal11—MeBmt1 can balance between polar exposure to improve solubility in water and intramolecular hydrogen bonding to stabilize a folded state via water ligands. The relevance of this new CycA conformer, as shown in this work, resides in its ability to rationalize the wide spectrum of biological activities observed for CycA, CycG, CycH, and CycE, and suggest that CycA may bind CypA via an “inversed” induced fit model.

Atomic view of aqueous cyclosporine A: unpacking a decades-old mystery

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