Fig. 1.

The closed state of the Met20 loop contains two interconverting substates. (A) Schematic of the hydride transfer reaction catalyzed by DHFR. Hydride transfer occurs from NADPH to DHF, yielding NADP⁺ and THF. The reaction proceeds by a stepwise mechanism: protonation of DHF from water precedes hydride transfer. The N5 nitrogen and C6 carbon of DHF are labeled. (B) and (C) $2m{F}_o-D{F}_c$ map (blue mesh; 0.7$\sigma$), $m{F}_o-D{F}_c$ (green mesh; +4.0$\sigma$), and refined model for a ecDHFR:NADP⁺:FOL structure at 290 K. (B) The ecDHFR complex adopts the Met20 closed conformation and two rotamer states can be modeled for Met20 (both shown in stick representation), accompanied by unmodeled density. The Bottom panel depicts how this electron density can be interpreted as a superposition of an “accessible” state that allows water into the active site and an “inaccessible” state that occludes water. (C) The region composed of Met20, Pro21, and Trp22 adopts two conformations marked by distinct backbone conformations between Pro21 and Trp22 (blue and red arrows). (D) Kernel density estimates of the Trp22-$\varphi$ dihedral from MD simulations in the context of a crystal lattice and a solvated water box, and a histogram of the Trp22-$\varphi$ dihedral in deposited structures of ecDHFR. The two states observed in (C) are shown with corresponding blue and red arrows, and the Inset structure indicates the Trp22-$\varphi$ dihedral. The $2m{F}_o-D{F}_c$ and $m{F}_o-D{F}_c$ maps shown in (B) and (C) are carved within 1.5 and 3 Å, respectively, of the indicated residues for clarity.