Methotrexate interaction with DHFR

Figure 12.28. Correlated motions of a carboxylate group from methotrexate and Arg57 of DHFR detected by NMR (a) Structure of an arginine-car-boxylate complex formed with symmetrical end-on interactions and (b) structure of methotrexate showing its interaction with the guanidine group of Arg57 of DHFR. (Reprinted with permission from Ref 133.)...

X-ray diffraction analysis of dihydrofolate reductase (DHFR), co-crystal-lized with methotrexate, has shed much light on the action of this inhibitor. This work, one of the earliest visualizations of a drug interacting with its receptor (Matthews etal., 1977), has since been refined to the remarkably clear resolution of 1.7 A (Bolin et al., 1982). A typical diagram of DHFR, its coenzyme (NADPH), and methotrexate is shown in Fig. 9.4. The enzyme depicted there is from Lactobacillus casei and the same authors also report on DHFR (with cocrystallized methotrexate) from the bacterium E. colt. However, they have not been able to co-crystallize methotrexate with DHFR from any vertebrate source. 

We have already used the interactions of methotrexate with dihydrofolate reductase (DHFR) several times within this text to illustrate some key aspects of enzyme inhibition. The reader will recall that methotrexate binds to both the free enzyme and the enzyme-NADPH binary complex but displays much greater affinity for the latter species. The time dependence of methotrexate binding to bacterial DHFR was studied by Williams et al. (1979) under conditions of saturating [NADPH], In the presence of varying concentrations of methotrexate, the progress curves for DHFR activity became progressively more nonlinear (Figure 6.14). The value of kobs from... 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

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