title: Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding.
abstract: 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.
authors.affiliation: Department of Chemistry, University of California, San Diego, La Jolla 92093.
authors.forename: C
authors.initials: C
authors.lastname: Bystroff
authors.forename: J
authors.initials: J
authors.lastname: Kraut
mesh_terms: D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D008958:Models, Molecular; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D001051:Apoenzymes; D008024:Ligands; D013762:Tetrahydrofolate Dehydrogenase