Figure 1.
DHFR subdomain and loop nomenclature.
Structure of DHFR complexed with NADPH (purple) and methotrexate (yellow) (pdbid 1RX3). Adenosine binding domain is shown in light blue while the loops domain is shown in wheat. The Met20 (red), F–G (blue), G–H (green) loops are labeled. The site of mutation (G121V) is indicated by a cyan sphere.
Figure 2.
Methotrexate binding traps G121V in the closed conformation.
(A) 1H–15N HSQC spectra of E:NADPH:MTX (black) overlain with the 1H–15N HSQC spectra of EG121V:NADPH:MTX (red). (B) The reduced change in chemical shift is calculated using the following formula and is subsequently plotted as a function of residue number: . The changes as a result of mutation (G121V vs. WT) are shown in black and a bona fide closed–occluded change are plotted in red [23]. The plot shows that the pattern of changes as a result of mutation can be attributed to differences in the local chemical environment and not large-scale structural change. The Met20 (residues 9–23), F–G (residues 117–131), and G–H (residues 142–149) loops are highlighted.
Figure 3.
G121V dependent changes in µs-ms motions.
Backbone residues with significant (>1.5σ) Rex, as determined by 15N CPMG relaxation experiments for E:NADPH:MTX (A) and EG121V:NADPH:MTX (C). For EG121V:NADPH:MTX, residues with calculated exchange rates are in pink while residues that could not be assigned because of line broadening are in red. Common residues that could not be assigned in either ternary complex are not included. These residues also mapped onto the structure using black spheres for E:NADPH:MTX (B) and pink or red spheres, denoting CPMG-based Rex or missing respectively, for EG121V:NADPH:MTX (D).
Figure 4.
G121V dependent changes in backbone and side-chain dynamics on the ps-ns timescale.
Changes in backbone S2 (A) and τe (B) order parameters determined from 15N relaxation experiments. Changes in side-chain S2axis (D) and τe, axis (E) order parameters determined from 2H relaxation experiments. Plotted values were obtained by subtracting mutant (EG121V:NADPH:MTX) from wild-type (E:NADPH:MTX) parameters with significant changes (>1.5σ) highlighted in red (backbone) and blue (side-chain). Significant changes are also mapped onto the structure using red spheres for backbone (C) and blue spheres for side-chain (F). Greens spheres denote residues that have significant changes in both backbone and side-chain order parameters. An area of 5 Å around the active site is highlighted in green.
Table 1.
Model-free analysis of tryptophan indole 15N relaxation within E:NADPH:MTX and EG121V:NADPH:MTX complexes.