CYP119 NMR and X-ray Analysis Suggest Unique Binding Modes for Similar Sized Ligands

CYP119 is a thermophilic enzyme found in the Archaebacteria Sulfolobus acidocaldarius. Several published crystal structures as well as NMR show a large degree of conformational flexibility on binding azole inhibitors such as imidazole and 4-phenylimidazole. The flexibility present in CYP119 on binding different size ligands is rare in bacteria, and provides a good model for the flexibility seen in the more promiscuous drug metabolizing CYPs. A central question is whether ligand binding stabilizes one of many conformational states in a heterogeneous mixture, or if an “induced fit” model where structural changes are unique for each ligand is more likely. We have previously used ¹³C- labeled methoxy phenylalanine NMR to show that CYP119 samples the substrate-free state in solution even when bound to smaller azoles^1,2. Further, we have found that methoxy-Phe153 exhibits an intermediate NMR peak on substochiometric concentrations of imidazole and phenyl imidazole ligands. The crystal structure of imidazole bound CYP119 exhibits a closed structure in which the FG helix closes down over the active site. In contrast the flexible FG helices and FG loop dramatically retract away from the active site upon phenyl-imidazole binding. We have explored ¹⁵N labeled phenylalanine NMR and found that CYP119 binding of 4-(4-bromo-phenyl)-1H-imidazole exhibits discrete chemical shifts in Phe153 and Phe162. In comparison, 4-(4-chloro-phenyl)-1H-imidazole and 4-(4-flouro-phenyl)-1H-imidazole bound enzymes exhibit chemical shifts similar to the phenyl-imidazole bound structure. X-ray crystal structures of CYP119 complexed with 4-(4-bromo-phenyl)-1H-imidazole or 4-(4-chloro-phenyl)-1H-imidazole show a dramatic 180° flip in the FG loop compared to the phenyl imidazole bound crystal structure. In contrast, the X-ray structure of CYP119 bound to 4-(4-flouro-phenyl)-1H-imidazole is largely similar to the phenyl imidazole bound structure. It is significant that we observe a discrete change in conformation rather than a gradual retraction on binding the bromine and chlorine phenyl imidazole derivatives. The increase in size from a fluorine to bromine substituent is enough to trigger a dramatic reorganization of the active site. Further, the discrepancy between NMR and X-ray data when a chlorine atom is present at this position suggests that similar ligands can reveal discrete binding modes in CYP119, not all of which are observable in crystal structures. (1) Lampe, J. N., Brandman, R., Sivaramakrishnan, S., and de Montellano, P. R. O. (2010) Two-dimensional NMR and all-atom molecular dynamics of cytochrome P450 CYP119 reveal hidden conformational substates. J. Biol. Chem. 285, 9594–9603. (2) Lampe, J. N., Floor, S. N., Gross, J. D., Nishida, C. R., Jiang, Y., Trnka, M. J., and Ortiz de Montellano, P. R. (2008) Ligand-Induced Conformational Heterogeneity of Cytochrome P450 CYP119 Identified by 2D NMR Spectroscopy with the Unnatural Amino Acid 13C- p-Methoxyphenylalanine. J. Am. Chem. Soc. 130, 16168–16169.