posted on 2024-03-16, 19:03authored byMiranda
N. Limbach, Edward T. Lindberg, Hernando J. Olivos, Lara van Tetering, Carlos A. Steren, Jonathan Martens, Van A. Ngo, Jos Oomens, Thanh D. Do
Our study reveals the underlying principles governing
the passive
membrane permeability in three large N-methylated
macrocyclic peptides (N-MeMPs): cyclosporine A (CycA),
Alisporivir (ALI), and cyclosporine H (CycH). We determine a series
of conformers required for robust passive membrane diffusion and those
relevant to other functions, such as binding to protein targets or
intermediates, in the presence of solvent additives. We investigate
the conformational interconversions and establish correlations with
the membrane permeability. Nuclear magnetic resonance (NMR) and cyclic
ion-mobility spectrometry-mass spectrometry (cIMS-MS) are employed
to characterize conformational heterogeneity and identify cis-amides relevant for good membrane permeability. In addition,
ion mobility selected cIMS-MS and infrared (IR) multiple-photon dissociation
(IRMPD) spectroscopy experiments are conducted to evaluate the energy
barriers between conformations. We observe that CycA and ALI, both
cyclosporines with favorable membrane permeabilities, display multiple
stable and well-defined conformers. In contrast, CycH, an epimer of
CycA with limited permeability, exhibits fewer and fewer stable conformers.
We demonstrate the essential role of the conformational shift from
the aqueous cis MeVal11–MeBmt1 state (A1) to the closed conformation featuring cis MeLeu9–MeLeu10 (C1) in facilitating membrane
permeation. Additionally, we highlight that the transition from A1 to the all-trans open conformation (O1) is specifically triggered by the presence of CaCl2. We also capture a set of conformers with cis Sar3–MeLeu4, MeLeu9–MeLeu10, denoted as I. Conformationally selected cIMS-MS and IRMPD data of [CycA+Ca]2+ show immediate repopulation of the original population distribution,
suggesting that CaCl2 smooths out the energy barriers.
Finally, our work presents an improved sampling molecular dynamics
approach based on a refined force field that not only consistently
and accurately captures established conformers of cyclosporines but
also exhibits strong predictive capabilities for novel conformers.