Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics

Abstract

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

Effect of Salts on the Conformational Dynamics of the Cytochrome P450 OleP

“…This finding is consistent with the experimental observation that high ionic strength favors closed-like conformations of the OleP-6DEB complex [ 7 , 11 ].…”

“…Therefore, because the electrostatic properties of the environment may affect the strength of intramolecular salt links, the dependence of the structural transitions of a protein on the presence of electrolytes in solution, as is the case of OleP, points out on a conformational dynamics, which is under the ionic tethering control. Then, as described for other P450s [ 27 , 28 , 32 ], we may expect that an increase of ionic strength that stabilizes the closure of OleP ( Figure 5 and Figure 6 and [ 5 , 7 ]), facilitating water expulsion from the active site ( Figure 7 b,c), and shifts the spin of the heme-iron to the high configuration ( Figure 2 and Figure S3 and [ 7 , 11 ]), may cause the dissociation of some internal salt links of the protein. Searching for intramolecular electrostatic interactions that may act as ionic tethers in OleP, we compared the open and closed structures of OleP-6DEB, and we identified four ionic contacts over a total of 17, which are broken along the open-to-closed transition ( Figure 9 , Table S5 ).…”

“…Equilibrium-binding experiments and X-ray structural analysis have shown that ionic strength alters the equilibrium between low- and high-spin populations of OleP bound to 6DEB: as ionic strength increases, the high-spin-state complex is stabilized, by enhancing OleP closure which allows the aglycone substrate to displace the sixth coordinating water molecule [ 5 , 7 , 11 ].…”

“…Equilibrium-binding experiments and crystallographic analysis previously reported have shown that the conformational dynamics of the substrate-bound OleP can be altered by variations of the ionic strength conditions. More specifically, a high-ionic strength environment (I ≥ 4 M) favors the full accumulation of the closed conformer in crystals, whereas in solution, an increase of ionic strength (up to I ≈ 3.5 M) causes a transition of the absorption spectra toward the high-spin state in the spin equilibrium [ 5 , 7 , 11 ]. Therefore, increasing the ionic strength shifts the equilibrium toward the high-spin closed form of the substrate-bound OleP, which is the less populated conformer in physiological conditions (I ≈ 0.2 M) [ 5 , 7 , 11 ].…”

“…More specifically, a high-ionic strength environment (I ≥ 4 M) favors the full accumulation of the closed conformer in crystals, whereas in solution, an increase of ionic strength (up to I ≈ 3.5 M) causes a transition of the absorption spectra toward the high-spin state in the spin equilibrium [ 5 , 7 , 11 ]. Therefore, increasing the ionic strength shifts the equilibrium toward the high-spin closed form of the substrate-bound OleP, which is the less populated conformer in physiological conditions (I ≈ 0.2 M) [ 5 , 7 , 11 ]. The way by which ionic strength influences the structural change of the substrate-OleP complex and whether the effect may be enhanced by specific cations remains unknown.…”