Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics

Abstract

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Figure 1

Structure of E. coli DHFR with bound NADPH and methotrexate (Protein Data Bank ID code 1rx3). The central portion of the Met-20 loop (residues 17–20) is colored green with the green sphere indicating the location of the Alexa 488 fluorophore (residue 18). The blue sphere represents the N terminus, which is the site of biotinylation. NADPH and methotrexate are colored brown and red, respectively.

Figure 2

Fluorescence titration of DHFR with methotrexate. The fraction of bound enzyme, (E.Mtx)/(E₀), is plotted versus the free ligand concentration, (Mtx-free). Data were obtained from the fluorescence quenching for both tryptophan (A) and Alexa (B). The curves were calculated by assuming a dissociation binding constant of 9.5 nM.

Figure 3

Stopped-flow traces for methotrexate binding to DHFR. The fluorescence, in arbitrary units, is plotted versus time, with the concentration of methotrexate (μM) given for each trace. The solid lines represent the least-squares fit of the data.

Figure 4

The observed first-order rate constants, determined from the data in Fig. 3, for the binding of methotrexate to DHFR are plotted versus the concentration of methotrexate. Here k₁ (●) is for the fast phase, and k₂ (○) for the slow phase.

Figure 5

The time course for the dissociation of methotrexate from the enzyme-methotrexate complex is shown for displacement by 700 μM (A) and 350 μM (B) trimethoprim. The solid lines are calculated by assuming a single exponential increase in fluorescence.

Figure 6

Three-dimensional representation of single molecules of DHFR (0.1 M sodium phosphate, pH 7.0, ≈25°C) as viewed by fluorescence microscopy. The vertical dimension is the fluorescence intensity in arbitrary units. National Institutes of Health image software was used to generate this plot. (Magnification: ×100.)

Figure 7

Representative trajectory for the blinking of single molecules of the DHFR-methotrexate complex. The fluorescence intensity, in arbitrary units, is plotted versus the time. The free methotrexate concentration was 5 nM.

Figure 8

Representative distribution of single-molecule reaction times for the transition from the low intensity state to the high intensity state. The number of events within a given range of reaction times is shown as a series of rectangles and plotted versus the reaction time, τ. The line assumes a single exponential distribution as described in the text. The free methotrexate concentration was 5 nM.