The solution structure of some cobalamins determined by NMR-restrained molecular modelling

The complete 1H and 13C NMR assignments of four cobalamins, namely nitrocobalamin, ammoniumcobalamin, sulphitocobalamin and glycylcobalamin, were made from 2D NMR experiments. Inter-proton distances were determined from nuclear Overhauser effects observed in the rotating-frame Overhauser enhancement (ROESY) spectra. These were incorporated as distance restraints in molecular dynamics (MD) and simulated annealing (SA) calculations using a previously derived force field for the molecular mechanics modelling of the cobalt corrins, to which were added new parameters for modelling the coordination sphere of Co(III) in these compounds. Consensus structures were obtained by overlaying 30 annealed structures obtained by MD/SA procedures with different run times, averaging the coordinates, and energy minimising. The inter-proton distance criteria were well preserved in the modelling of all four compounds during a 400 ps MD simulation at 300 K. All four ligands experience restrictions to free rotation about the cobalt-ligand bond, principally due to close van der Waals contact with the C19 proton. During dynamics simulations, the mean conformation adopted by the A and C pyrrole rings is an envelope whilst that of the B and D rings is a half chair. The B and C pyrrole rings undergo greater distortions than the A and D rings. The increased rigidity in the ‘western’ half of the molecule is probably a consequence of the direct linkage of the A and D pyrrole rings. In solution, the corrin ring undergoes a ‘breathing motion’ in which C5, C10, and C15 oscillate from above to below the mean corrin plane. The flexibility of the corrin ring increases in the order NO2CblzNH3CblC!SO3CblK!GlyCbl which indicates that in these complexes bulkier ligands tend to induce greater distortions in the macrocycle because of their more extensive interactions with the corrin. The 5,6- dimethylbenzimidazole (dmbzm) ligand oscillates about its mean position in which its projection onto the mean plane through the corrin lies virtually along the C5/Co/C15 vector, constrained by the downward pointing b and d side chains of the corrin. The motions of the side chains in these complexes are very similar to those reported previously for other cobalt corrins and not all possible locally minimum energy conformations are visited. The ribose of the nucleotide loop oscillates between a 20-endo conformation and an envelope conformation; in its mean position it is in a 30-endo conformation. A comparison of the consensus structures and the available crystal structures of NO2Cbl and SO3CblK show they are very similar. In NO2Cbl minor differences occur in the disposition of the NO2 group relative to the corrin ring, and the c, d, and f side chains. In SO3CblK some differences occur in the disposition of the c and e side chains relative to the corrin ring, and the f side chain relative to the ribose ring, as well as the ribose ring itself. This work shows that nOe-restrained molecular dynamics simulations provide a reliable method for investigating the solution structures of the cobalt corrins. Moreover, since the present results and those reported previously indicate that the solution structure and solid state structure of these compounds are very similar, these methods provide a means of exploring the structure of those cobalt corrins that fail to crystallise. q 2004 Elsevier B.V. All rights reserved.