J. Gready, P. Cummins, U. Singh, K. Ramnarayan
Division of Biochemistry and Molecular Biology,
John Curtin School of Medical Research, Canberra, Australia
Cummins PL, Ramnarayan K, Singh UC, Gready JE
Journal of the American Chemical Society 1991,113,8247
Molecular-dynamics free-energy perturbation study on the relative affinities of the binding
of reduced and oxidized NADP to dihydrofolate-reductase
The free energy perturbation (FEP) method, implemented within the molecular dynamics (MD) simulation scheme, has been used to
investigate the difference between the binding of reduced and oxidized nicotinamide adenine dinucleotide phosphate (NADP) cofactor to
Escherichia coli dihydrofolate reductase (DHFR) (binary complex) and E. coli DHFR bound to the substrate dihydrofolate (ternary complex)
in aqueous solution. The FEP results for the binary complex predict relative equilibrium binding constants for the reduced and oxidized forms
of NADP to E. coli DHFR in good agreement with the available experimental data, suggesting that reduced NADP binds some 10(2) times
more strongly than oxidized NADP. The FEP results also predict reduced NADP in the ternary complex with dihydrofolate to bind more
strongly than oxidized NADP. However, this differential is calculated to be 10(2)-10(3) times greater than in the binary complex. Although
there is no direct experimental information for binding in the active ternary complex with which to compare these results, available results
are discussed in the context of the molecular form of active complexes seen kinetically compared with those in the theoretical simulations.
The question of the influence of the choice of initial enzyme coordinates and configuration space sampling in these simulations is also
discussed. The stronger binding affinity of reduced NADP and the differences between NADP binding strengths computed for the binary and
ternary complexes are correlated with solvation effects and structural differences between the complexes. Analysis of the MD structures and
available crystallographic data suggests that the positioning of a mobile loop (the "Met-20 loop") plays a key role in determining the relative
cofactor binding strengths.
Please note that the format of the frcmod file is an old version, with
four letter atom identifiers. To use this file, it will require modification
to two letter atom labels.
HF/6-31G* RESP charges used and are non-integral due to derivation procedure via molecular fragments.
Parameters were developed originally for use with AMBER3.1 (scee=2.0)
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