Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode

Abstract

Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH-dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP(+) has been determined at 1.15 angstroms resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and pyrophosphate group (PP(i)) extending toward the outer pore. The ribonicotinamide exhibits anti conformation as in chromosomal DHFR complexes. The relative orientation between the PP(i) and the nicotinamide ribose differs from that observed in chromosomal DHFR-NADP(+) complexes. The coenzyme displays symmetrical binding mode with several water-mediated hydrogen bonds with the protein besides ionic, stacking, and van der Waals interactions. The structure provides a molecular basis for the observed stoichiometry and cooperativity in ligand binding. The ternary model based on the present structure and the previous R67 DHFR-folate complex provides insight into the catalytic mechanism and indicates that the relative orientation of the reactants in plasmid DHFR is different from that seen in chromosomal DHFRs.

Figure 1.

Cofactor binding and stoichiometry. (A) The enzyme is comprised of four subunits (1–4) shown in gray and black. The simultaneous binding of two cofactor molecules (CPK model without adenosine 2′-phosphate portion) on the front or rear side of the active site pore results in steric clash, primarily between the ribose groups. (B) The protein portions comprised of residues Val66, His67, Ile68, and Tyr69 from four subunits bordering the central active site pore are shown as black and gray ribbons. The paired cofactor molecules that approach each other from opposite sides (indicated by arrows that align with a twofold symmetry axis) exhibit minimal stacking of the nicotinamide rings. Vertical bars align with a twofold axis, and the symmetry-related His67 side-chains are shown as van der Waals surfaces. The central dot shows another twofold axis passing through the plane of the paper. (C) Schematic diagram representing the possible modes of cofactor binding. Left and right of the vertical bar denote the front and rear portions of the pore as viewed down one of the two short axes of the active site. There are two binding positions in each half of the pore. The sites represented as horizontal lines are labeled A and B for one half of the pore and C and D for the other half of the pore (top panel on the left). The center of the pore is depicted as a vertical bar. The three lower panels on the left show possible binding modes without steric hindrance. The bottom panel on the left shows one bound NADP⁺ molecule in site A (see text for details). It can also be bound in either site B or C or D (data not shown). The right part of the figure shows different binding modes that are precluded due to stereochemical clashes between the cofactors. It may be noted that binding site A stacks on site D and clashes with site C but is not shown in the figure for clarity (the same applies to site B). The extent of stacking of ligands is larger in the R67 DHFR–folate complex than in the holoenzyme complex.

Figure 2.

Omit difference Fourier map and environment of the bound cofactor. F_o − F_c omit map was computed using protein atoms and solvent molecules, excluding those in the active site pore. (A) The nicotinamide ring is superimposed on the map contoured at ∼2.5σ. (B) The density for nicotinamide ribose (∼1.5σ) was supportive for an anti conformation about the glycosidic bond. (C) The PP_i moiety was seen at ∼3.0σ level (gray). The anomalous difference Fourier map (20–1.15 Å resolution) showed distinct and strong peaks (blue) corresponding to the PN and PA atoms of the cofactor. (D) Schematic diagram depicting direct and water-mediated (single or double) interactions (distances ≤3.3 Å) between the cofactor and the enzyme. The cofactor atoms are shown as sticks. The atoms belonging to the four subunits are in blue, cyan, green, and red. The dashed lines indicate hydrogen bonds.