Long-Range Distance Measurements of Protein Binding Sites by Rotational-Echo Double-Resonance NMR

Abstract

Figure 1. (Left) CR trace of 5-enolpyruvylshikimate-3-phospate synthase, based on the X-ray diffraction results of ref 16, showing the approximate locations of W172 (upper domain) and W337 (lower domain) of the double mutant W289Q;F172W. The side chain of F172 in the wild-type structure can be modified to tryptophan with retention of the wild-type rotameric state and only slight adjustment to remove van der Waals contacts with nearby residues. The F-F distance in this structure is 38 A. (Right) CR trace of a model of the EPSPS ternary complex consistent with 19 F- 31 P REDOR distance measurements (cf. Figure 3). Ligands were docked into the X-ray structure on the basis of the results of REDOR studies involving complexes of specifically 13 C- and 15 N-labeled protein and ligands (refs 17-21). The hinge was identified by searching for residues in the connecting strands that were not part of a secondary structural element in either the upper or lower domain. Torsions in the two hinge strands in EPSPS were manually adjusted to bring the two protein domains into proximity. Ligand position and hinge torsions were adjusted iteratively to accommodate the P-F distances measured in this study and C-N, C-P, and N-P distances measured previously. 17-21 The F-F distance in this model is 21 A. The F -P distances for W337-S3P, W337-Glp, W172-S3P, and W172-Glp in the model are 18, 13, 7, and 11 A, respectively.