*2.6. All-Atom MD Simulations Confirm Di*ff*erences in the Positioning of the Globular Domain on the Membrane Between CYP 2C9 and CYP 2C19*

The positioning of the globular domain with respect to the membrane was analyzed by calculating the heme-tilt angle in addition to the angles and distances computed for the CG simulations. The computed values are given in Table 2. The normalized angle and distance distribution plots characterizing the position of the globular domain observed in the simulations are shown in Figure 4. The simulations of CYP 2C9 showed some readjustment in the orientations from the starting configurations in which the β angle increased from about 112◦ to 120–127◦, corresponding to remaining in class A in two cases (for the apoprotein and for the holoprotein with a substrate, the drug flurbiprofen, bound in the active site) and transitioning to class A/B in one case (SIM3), in which the globular domain structure was slightly less stable (see Figures S4 and S5). Concomitantly, the heme-tilt angle increased from 30–34◦ to 40–43◦. The average axial distance of the globular domain CoM from the membrane CoM increased by 0.5 to 3 Å during the simulations. Compared to previous simulations conducted with the GAFF lipid force field [22], in which the globular domain CoM distance from the

bilayer center decreased from 39.5 ± 2.5 Å (from CG simulations) to 34.1 ± 1.0 Å during AA simulations, the globular domain was less immersed in the membrane, facilitating the slightly lower observed values of the α and β angles. These differences can be attributed in large part to differences in both the protein and the lipid force field used. Our studies have shown that the force field used in the current work produces results in better agreement with experimental data for simulations of CYP-membrane systems, including excellent agreement with linear dichroism measurements of the heme-tilt angle of CYPs in Nanodiscs [25–27].

**Figure 4.** Plots of the distance and angle distributions defining the positioning of the globular domain with respect to the membrane in CG and AA MD simulations of CYP 2C9 (**A**) and CYP 2C19 (**B**). The globular domain of CYP 2C9 tended to be more immersed in the membrane than CYP 2C19 and the two adopted distinct orientations: class A (angle β < 125◦) for CYP 2C9, and class B (angle β > 130◦) for CYP 2C19.

The starting structures of the two AA models of CYP 2C19 in the membrane (for the apoprotein and for the holoprotein with the inhibitor 0XV bound in the active site) varied slightly in the heme-tilt angle: 52◦ in the structure from CG:S1 and 46◦ in the structure from CG:S2. The distance of the CoM of the globular domain to the bilayer center was also different in the two starting structures: 47 Å in CG:S1 and 50 Å in CG:S2. During all three AA simulations, the globular domain fluctuated around the starting position. The angles ranged between 95 ± 5◦ to 106 ± 4◦ for α and 136 ± 7◦ to 149 ± 5◦ for β (corresponding to class B), with an increase during the simulations of the heme-tilt angle to 55 ± 7◦ to 61 ± 5◦. The axial distance of the CoM of the globular domain from the bilayer center stayed constant at about 46 ± 2 Å in the AA simulations, independently of whether a ligand was present (SIM2) or the TM-helix was truncated (SIM3).

In summary, the differences in the orientations and interactions of the two proteins (CYP 2C9 and CYP 2C19) observed in the CG simulations were maintained and, in some cases, became more pronounced during the AA simulations. Comparison of simulations of the apo- and holo- forms of the proteins indicate that the orientation of the proteins in the membrane was not significantly affected by the presence of a compound in the active site.
