The crystal structure of a tetrahydrofolate-bound dihydrofolate reductase reveals the origin of slow product release

Abstract

Dihydrofolate reductase (DHFR) catalyzes the stereospecific reduction of 7,8-dihydrofolate (FH2) to (6s)-5,6,7,8-tetrahydrofolate (FH4) via hydride transfer from NADPH. The consensus Escherichia coli DHFR mechanism involves conformational changes between closed and occluded states occurring during the rate-limiting product release step. Although the Protein Data Bank (PDB) contains over 250 DHFR structures, the FH4 complex structure responsible for rate-limiting product release is unknown. We report to our knowledge the first crystal structure of an E. coli. DHFR:FH4 complex at 1.03 Å resolution showing distinct stabilizing interactions absent in FH2 or related (6R)-5,10-dideaza-FH4 complexes. We discover the time course of decay of the co-purified endogenous FH4 during crystal growth, with conversion from FH4 to FH2 occurring in 2–3 days. We also determine another occluded complex structure of E. coli DHFR with a slow-onset nanomolar inhibitor that contrasts with the methotrexate complex, suggesting a plausible strategy for designing DHFR antibiotics by targeting FH4 product conformations.

Time-resolved x-ray crystallography capture of a slow reaction tetrahydrofolate intermediate

“…We conjecture that it acts as a clamp that is partly responsible for the slowdown of tetrahydrofolate to dihydrofolate oxidation. The Met20 loop appears to be partially disordered for residues Glu17-Asn18-Ala19 in contrast to the clearly traceable conformations of the end-state complexes with tetrahydrofolate (PDB: 6CW7) or dihydrofolate (PDB: 6CXK) that were visible in our earlier report 14 . On the other hand, Met20 and Pro21 are clearly visible and are quite close to their conformation in the dihydrofolate complex.…”

“…The current intermediate time point complex was obtained using the same crystal growth condition as in our previously reported binary tetrahydrofolate complex (PDB: 6CW7) 14 . Briefly, as-purified eDHFR was crystallized by sitting drop vapor diffusion using a 1:1 v/v mixing of 20 mg ml −1 DHFR solution in 13.3 mM Tris at pH 8, 16.7 mM HEPES at pH 7.3, 33.3 mM NaCl, and 0.67 mM DTT with the reservoir solution containing 0.1 M MES, at pH 6.5, with 30% w/v PEG 3350, and 0.4 M MgCl 2 .…”

“…Mixed drops of 0.8 μ l were equilibrated over a reservoir solution of 50 μ l on a MRC 2-well plate (Hampton Research) and incubated at 20 °C in the dark. A single crystal per dataset was harvested at 3 days after setting up the crystallization drops (as opposed to 2 days for the tetrahydrofolate complex or 2 weeks for the dihydrofolate complex in our earlier report) 14 and then cryoprotected with LV CryoOil and flash-frozen in liquid N 2 .…”

“…To the best of our knowledge, time-resolved crystallography has almost exclusively been applied on time scales of picoseconds to seconds. Here, using traditional cryocrystallography at a synchrotron source without rapid mixing or use of a laser pulse, we recently discovered the slow oxidative decay of tetrahydrofolate to dihydrofolate in the enzyme bound crystalline form at room temperature, with the critical transition occurring 2–3 days after the crystallization setup 14 . We present an analysis of the third day's crystal structure to estimate the putative intermediate's geometry and population relative to dihydrofolate and tetrahydrofolate.…”

Substrate binding and turnover modulate the affinity landscape of dihydrofolate reductase to increase its catalytic efficiency

“…1A). Five intermediates have been identified in the steady-state catalytic cycle 22 and characterized by X-ray crystallography [23][24][25][26][27] and NMR 18,20,21,28,29 : E:NADPH, E:NADPH:DHF, E:NADP + :THF, E:THF, and E:NADPH:THF ( Fig. 1B).…”