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The crystal structure of unliganded dihydrofolate reductase (DHFR) from
Escherichia coli has been solved and refined to an R factor of 19% at 2.3-A
resolution in a crystal form that is nonisomorphous with each of the previously
reported E. coli DHFR crystal structures [Bolin, J. T., Filman, D. J., Matthews,
D. A., Hamlin, B. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662;
Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277].
Significant conformational changes occur between the apoenzyme and each of the
complexes: the NADP+ holoenzyme, the folate-NADP+ ternary complex, and the
methotrexate (MTX) binary complex. The changes are small, with the largest about
3 A and most of them less than 1 A. For simplicity a two-domain description is
adopted in which one domain contains the NADP+ 2'-phosphate binding site and the
binding sites for the rest of the coenzyme and for the substrate lie between the
two domains. Binding of either NADP+ or MTX induces a closing of the
PABG-binding cleft and realignment of alpha-helices C and F which bind the
pyrophosphate of the coenzyme. Formation of the ternary complex from the
holoenzyme does not involve further relative domain shifts but does involve a
shift of alpha-helix B and a floppy loop (the Met-20 loop) that precedes alpha
B. These observations suggest a mechanism for cooperativity in binding between
substrate and coenzyme wherein the greatest degree of cooperativity is expressed
in the transition-state complex. We explore the idea that the MTX binary complex
in some ways resembles the transition-state complex.
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