Fig 1.
Overview of experimental design and results.
The figure was created with BioRender.com.
Table 1.
Data collection and refinement statistics.
Fig 2.
A ribbon diagram of WbDHFR (cyan) shows the bound complex with folate and NADPH (yellow) with the atom labels of folate and NADPH.
(A). An amino acid sequence alignment of eight prokaryotic and eukaryotic DHFRs is shown (B). Amino acids corresponding to the Met20 loop are highlighted in red in A and in a red rectangle in B. Sequences of DHFR orthologs were obtained through UniProt and aligned with the multiple sequence alignment tool “ClustalW” within Jalview. Amino acids with similar chemical characteristics were colored using the “ClustalX” color scheme. Amino acids observed to make hydrogen bonds with either folate or NADPH are labeled “F” and “N”, respectively. The first amino acid residues for Hs, Mm and Sa DHFR are denoted as #2 because these sequence entries are derived from proteins purified from the organisms rather than cloned cDNA.
Fig 3.
Determination of KDs of folate (A) and NADPH (B) with WbDHFR using tryptophan fluorescence measurements. Approximately 400 nM of Ni-NTA purified WbDHFR was titrated with folate (0–4 μM; A) and with NADPH (0–10 μM; B) in separate experiments. Fluorescence intensity (ex. 290 nm, em. 340 nm) of the sample was recorded using a Fluoromax-4 spectrofluorimeter. Three trials were normalized, averaged, and the data were fitted to the Morrison equation using Kaleidagraph [41].
Fig 4.
WbDHFR (cyan) with amino acids involved in folate (A, B) and NADPH (C, D) binding are shown as light blue sticks. Three-dimensional representations (A and C) were generated in PyMOL while the two-dimensional images (B and D) were made in Maestro. Symbols used in B and D are described in E. The PDB entry 8E4F was used to generate these figures.
Table 2.
The sequence identities and RMSD values for WbDHFR bound to NADPH and folate compared with 13 other DHFR structures.
Note: All structures are of the enzyme bound to cofactor NADPH and an antifolate except for SmDHFR (PDB 3VCO).
Fig 5.
Results of molecular docking of folate and antifolates into the WbDHFR active site using AutoDock Vina.
Hydrogen bonds between the co-crystallized folate (A) and WbDHFR (PDB: 8E4F) are denoted as yellow dashes. Green dashes indicate potential hydrogen bonds between the docked antifolates and WbDHFR. In panel A, co-crystallized folate (yellow) and docked folate (orange) exhibit comparable conformations (See Fig A in S1 Text for structures). Larger inhibitors that resemble folate, i.e. methotrexate (green; B) and aminopterin (pink; E) and smaller antifolates pyrimethamine (magenta; C), trimethoprim (dark red, D), and trimetrexate (yellow, F), are shown. The energy scores from AutoDock Vina and Schrödinger Glide for docked ligands are shown in G.
Fig 6.
Interaction fractions depicted for WbDHFR and various antifolate types.
Antifolates with longer (A), shorter (B), or moderate length linkers (C) were grouped to show hydrophobic binding patterns. Residues interacting with the antifolate are listed on the x-axis; interaction fraction on the y-axis describes the fraction of time each interaction is observed in the simulation (fraction of 1.0 = interaction is observed in 100% of snapshots). In cases where the interaction fraction is >1.0, the residues (or atoms) may be involved in more than one interaction. Common interactions are color-coded including hydrogen bonds (green), hydrophobic contacts (purple), ionic bonds (pink), and water bridges (blue) (D). Comparison of interaction fractions shows that the long linker antifolates make more hydrophobic contacts.
Fig 7.
The folate binding sites of the NADPH and folate bound DHFR structures from Wb (cyan; PDB 8E4F) and human (green; PDB 2W3M) were aligned using PyMOL.
Key differences in the binding pocket are shown.