NMR characterization of the ligand dependent dynamic changes in Cytochrome P450s

Cytochrome P450s are a large superfamily of enzymes in humans with little sequence homology, but a conserved fold. A characteristic feature of P450s is their ability to recognize and detoxify by mono-oxygenating a large number of drug ligands with diverse physico-chemical properties in humans. This is attributed to the flexibility of their substrate binding region. CYP101A1, the first solved structure of any cytochrome P450, is a bacterial enzyme that has been used as a model for P450s to study the binding and catalytic events. Recently, our laboratory in collaboration with other groups used neutron scattering spectroscopy to detect the presence of collective functional motions throughout the structure of this protein, that dynamically create access to the catalytic site for the substrate (1). However, the exact nature of these motions in terms of the range of timescales and identity of specific residues involved could not be determined. I sought to characterize a subset of these motions by studying the slow timescale dynamic changes of CYP101A1 when binding to a diverse set of ligands. Using amide exchange experiments monitored by 1H-15N two-dimensional solution NMR spectroscopy, dynamics were characterized at the residue level with 4 different ligands, including both substrates and inhibitors of assorted sizes and chemical properties. Comparing the dynamic changes between different ligands and the ligand free form showed surprising results. CYP101A1 exhibits differential dynamics for binding various ligands even though they all have the same affinity to the enzyme. Large ligands such as the anti-fungal drug ketoconazole shows increased dynamics while the much smaller substrate camphor shows highly reduced dynamics, pointing to a distinct enthalpy-entropy compensation mechanism. Moreover, these dynamic changes are observed throughout the protein and not just limited to regions in and around the active site, arguing that dynamics of the protein as a whole should be considered to properly understand ligand binding by this class of enzymes.