Fig. 5
Superposition of the FH4 complex with FH2 and FH4 analog complexes. The current FH4, FH2, and reported eDHFR complexes and the reported 5-formyl-FH4 complex along with their occluded Met20 loop conformations, Phe31 residues and the corresponding ligands are colored in green, magenta, and yellow, respectively. All other structures from PDB IDs: 1DYJ (ddFH4)45, 5CCC (ddFH4:NADP+)12, 1RF7 (FH2)24, 4PDJ (FH2:NADPH)20, and 4PSY (folate:NADP+)20 are colored gray. The red dashed circle indicates proposed π–π interactions between Phe31 and the ligand benzoyl groups that adopt two distinct orientations depending on the bound ligands. FH4 and 5-formyl-FH4 belong to one cluster in contrast to ddFH4, FH2, and folate, while the Phe31 side chains stay in nearly the same position in all aligned structures. The Met20 loops are categorized into three general conformational states, closed, partially closed, and occluded. Only the closed conformations can structurally accommodate the nicotinamide group of the NADP(H) cofactor entering the active site
