FIGURE 3.

Stereo overlays of the structures of wild type (PDB code 1JA1) (green), 147CC514 (gold), and the binary complex of reduced 147C/C514-NADP⁺ (gray). For clarity, only the isoalloxazine ring-ribityl portions of flavin cofactors are shown. A, comparison of wild type (green) and 147CC514 (gold with red labels). The NADP⁺ structure is that found in the wild type structure, because the cross-linked 147CC514 structure lacks bound NADP⁺. Residues labeled in black are those superimposed in both structures. The loop containing residues Gly⁶³¹–Asn⁶³⁵ (Asp⁶³² loop) in the structure of 147CC514 is extended down toward where the indole ring of Trp⁶⁷⁷ lies. This loop movement results in the guanidine group of Arg⁶³⁴ making hydrogen bonds with both the side chain and main chain amide nitrogens of Thr¹⁷⁷. Furthermore, the side chain of Asp⁶³² flips down and occupies the space where the ribityl-pyrophosphate moiety of NADP⁺ would ordinarily bind, causing steric hindrance as well as charge repulsion between the NADP⁺ pyrophosphate and Asp⁶³² carboxylate, preventing NADP⁺ from binding to 147CC514. B, overlay of the structure of NADP⁺-free 147CC514 (gold) and that of NADP⁺ bound disulfide-reduced 147CC514 (gray). Upon binding of NADP⁺, the Asp⁶³² loop of 147C/C514 is pushed further toward the FMN domain by as much as 6 Å for the Ala⁶³³ Cα atom and 4 Å for the Asp⁶³² Cα, making room for NADP⁺ to bind. Arg⁶³⁴ makes a salt bridge with the C-terminal carboxylate of Ser⁶⁷⁸. As shown in A, Asp⁶³² of 147CC514, which is located in the middle of the Asp⁶³² loop, may impair NADP⁺ binding by both steric hindrance and charge repulsion. C, comparison between wild type (green) and 147C/C514-NADP⁺ (gray). Both the Asp⁶³² loop and NADP⁺ conformations are different between these two structures; an extended NADP⁺ conformation is found in the wild type structure (green carbons with red oxygen and phosphorus atoms), although a more compact, folded conformation is observed in the mutant structure (gray carbons).