Methotrexate ring flip in DHFR

C. Verma, S. Fischer et al.
 J. Phys. Chem., 1996, vol.100, p.2510

NMR spectroscopy had been used to examine the dynamics of difluoro-methotrexate in its complex with dihydrofolate reductase (DHFR) from Lactobacillus Casei (Birdsall et al, Biochemical J., 1984, vol.217, p.659). It had been found that the benzoyl ring rotates at a microsecond timescale and the barrier to this rotation is characterized by an activation enthalpy of 11.5 kcal/mol.
  This process was simulated with the Conjugate Peak Refinement (CPR) method, yielding a computed energy barrier of 11.3 kcal/mol.  The corresponding barrier for methotrexate was computed to be 5.0 kcal/mol.

The reaction is characterized by an asynchronous transition of two dihedral angles adjacent to the benzoyl ring (coloured red and rotating in the image) gated by 4 hydrophobic residues, Leu19, Phe30, Phe49 and Pro50 (coloured blue in the image and shown anticlockwise; also shown in bottom right is Leu27). The gating is two-fold and is caused successively by the Leu19-Phe49 and Phe30-Pro50 pairs). The transition state has the ring almost perpendicular to its ground state conformation and lying such that one side of the ring is between Leu19 & Phe30 while the other side is between Phe49 & Pro50.

The perturbations to the protein matrix extend upto 8A from the rotating ring; the active site expands by a maximum of 1.6A. The methodology has been used to successfully reproduce the experimental barriers for both the ternary (with cofactor NADPH) and binary complexes of di-fluoromethotrexate bound to DHFR and additonally to calculate the same parameters for methotrexate for which these parameters could not be determined experimentally.

The work outlined above illustrates the success of simulations in studies where direct quantitative comparison with experiment is possible. Simulation also plays a powerful role in supplementing or interpreting experimental data, where quantitative measures are not always available or possible.

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