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Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA

Reprint requests to: Peter E. Wright, Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: wright{at}scripps.edu; fax: (858) 784-9822.

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. ¹H^N, ¹⁵N, and ¹³C resonance assignments have been made for the ternary complex with folate and oxidized NADP⁺ cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of ¹H^N and ¹⁵N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide–ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.

Keywords: Dihydrofolate reductase; Met 20 loop; conformational change; enzyme mechanism

Abbreviations: NMR, nuclear magnetic resonance • HSQC, heteronuclear single quantum coherence • DHFR, dihydrofolate reductase • NADP⁺, nicotinamide adenine dinucleotide phosphate • NADPH, reduced nicotinamide adenine dinucleotide phosphate • DHF, 7,8-dihydrofolate • THF, 5,6,7,8-tetrahydrofolate • DHNADPH, 5,6-dihydro NADPH

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