1H and 13C-NMR and molecular dynamics studies of cyclosporin a interacting with magnesium(II) or cerium(III) in acetonitrile. Conformational changes and cis-trans conversion of peptide bonds

Abstract

Cyclosporin A (CsA) is an important drug used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is related to the inhibition of T-cell activation through binding with the proteins Cyclophilin (Cyp) and, subsequently, Calcineurin (CN). In the complex with its target (Cyp), CsA adopts a conformation with all trans peptide bonds and this feature is very important for its pharmacological action. Unfortunately, CsA can cause several side effects, and it can favor the excretion of calcium and magnesium. To evaluate the possible role of conformational effects induced by these two metal ions in the action mechanism of CsA, its complexes with Mg(II) and Ce(III) (the latter as a paramagnetic probe for calcium) have been examined by two-dimensional NMR and relaxation rate analysis. The conformations of the two complexes and of the free form have been determined by restrained molecular dynamics calculations based on the experimentally obtained metal-proton and interproton distances. The findings here ratify the formation of 1:1 complexes of CsA with both Mg(II) and Ce(III), with metal coordination taking place on carbonyl oxygens and substantially altering the peptide structure with respect to the free form, although the residues involved and the resulting conformational changes, including cis-trans conversion of peptide bonds, are different for the two metals.

SCHEME 1

Chemical structure of the CsA molecule.

FIGURE 1

Comparison of free CsA conformation obtained from experimental data with those at various stages of the MD simulation in acetonitrile. Color codes are the following: best structure resulting from DYANA program (blue), after energy minimization (EM) in vacuo (cyan), after EM in acetonitrile (green), brought to 298 K (gold), after 100 ps of MD at 298 K (magenta).

FIGURE 2

(A) Comparison of 1D spectra of free CsA (bottom), CsA + 0.5 eq. Mg(II) (middle), and CsA + 1 eq. Mg(II) (top); (B) comparison of 1D spectra of free CsA (bottom), CsA + 0.5 eq. Ce(III) (middle), and CsA + 1 eq. Ce(III) (top).

FIGURE 3

Chemical shift variations (Δδ = δ(complex) − δ(free)) of carbonyl carbon, α-carbon, and α-proton signals of (A) the CsA-Mg(II) complex and (B) the CsA-Ce(III) complex in acetonitrile at 298 K with respect to the free form.

FIGURE 4

Plots of the chemical shift variations as a function of T for the mobile protons of the CsA-Mg(II) complex, with the temperature coefficients obtained by the fitting.

FIGURE 5

(A) Part of the ROESY spectrum of the CsA-Mg(II) complex, showing the crosspeaks between Hα of MeLeu⁹ and Hα of MeLeu¹⁰, and between Hα (1 and 2) of Sar³ and Hα of MeLeu⁴; (B) part of the ROESY spectrum of the CsA-Ce(III) complex, showing the crosspeaks between Hα of MeLeu⁹ and Hα of MeLeu¹⁰, and between Hα of Val⁵ and Hα of MeLeu⁶.

FIGURE 6

Best four structures of the CsA-Mg(II) complex (blue) superimposed with the one obtained after minimization (red) (A); comparison between the best structures obtained for free CsA (yellow), for the CsA-Mg(II) (blue), and the CsA-Ce(III) complex (cyan) (B); best four structures of the CsA-Ce(III) complex (blue) superimposed with the one obtained after minimization (red) (C). In panels A and C, the metal and its coordinating oxygens are shown as spheres.

FIGURE 7

Snapshots taken during the MD simulation of the CsA-Mg(II) complex in water (md2), showing the octahedral coordination of the Mg²⁺ ion with the carbonyl oxygens of residues 1, 4, 5, and 6 and with the oxygens of two water molecules (W1590 and W1592). The figure also shows the H-bond between the amide proton of Abu² and the carbonyl oxygen of MeVal¹¹, and the H-bonds between the oxydril oxygen of MeBmt¹ side chain and the hydrogen of water W1592, and between the corresponding oxydril proton and the oxygen of water W549.

FIGURE 8

Mg(II)-O(W1590) and Mg(II)-O(W1592) distances during the MD simulation of the CsA-Mg(II) complex in water.

FIGURE 9

Electrostatic potential surface of free CsA (A), the CsA-Mg(II) complex (B), and the CsA-Ce(III) complex (C); positively charged regions are shown in blue, negatively charged ones in red, and hydrophobic ones in white/gray. For each structure two views are shown, rotated by 180° with respect to one another.