Dynamics of the Dihydrofolate Reductase-Folate Complex: Catalytic Sites and Regions Known To Undergo Conformational Change Exhibit Diverse Dynamical Features

Backbone and tryptophan side-chain dynamics of uniformly ¹⁵N-labeled Escherichia coli dihydrofolate reductase were determined for the binary folate complex. The ¹⁵N T₁ and T₂ relaxation times and {¹H}-¹⁵N heteronuclear NOEs were measured for 118 protonated backbone nitrogen atoms. The generalized order parameter (S²), the effective correlation time for internal motions (τ_e), and the contribution to spin-spin relaxation through 15N exchange broadening (R_ex) were determined for each residue by model-free analysis. Back-calculation of the relaxation rates for each resonance showed that the calculated dynamical parameters accurately predict the experimental data. Diverse dynamical features were evident in the DHFR backbone. Six sites exhibited order parameters significantly below the weighted mean S² value (for the complex) of 0.81 ± 0.002: residues G67 and D69 of the adenosine binding domain, and “hinge” residues K38 and V88, exhibited low S² (0.29 ≤ S² ≤ 0.6) and high re values (700 ps ≤ τ_e ≤ 2 ns), as did sites within the βA-αB loop and the βF-βG loop. Thus, large amplitude backbone motions, on the picosecond and nanosecond time scales, occurred at regions implicated in transition-state stabilization and in ligand-dependent conformational change. Significant R_ex values (≥1 s^-1) were determined for 45% of assigned resonances, many of which arise from residues surrounding the folate binding site. The mean S² values of the occupied folate binding site and the unoccupied NADPH binding site were similar, indicating the backbone of the latter is at least as conformationally restricted as that of the occupied folate site. We conclude that the observed time-dependent structural fluctuations of the binary complex are in fact associated with catalytic properties of the molecule.