*2.4. CG Simulations of Chimeric Mutant Models Show Sequence Di*ff*erences in the Interfacial Residues A*ff*ect the Protein-Membrane Interactions and Globular Domain Orientations*

To identify the residues contributing to the differing position of the globular domain in the membrane, structures of chimeric mutants of CYP 2C9 (mt2C9) and CYP 2C19 (mt2C19) were prepared by swapping residues mainly at the membrane interface in the linker (G46D), β-strand 1–2 (K72E and P73R), the B–C loop (I99H), and the F'–G' helices (S220P and P221T) (see Table S1 and Figure 1). In CG simulations of mt2C9, two out of five trajectories converged to a CYP 2C19-like orientation (class B), one converged to an intermediate orientation (class A/B), and two converged to the same orientation as wild-type CYP 2C9 (class A). In CG simulations of mt2C19, two out of five trajectories converged to a CYP 2C9-like orientation (class A), one to an intermediate orientation (class A/B), and two retained wild-type CYP 2C19 orientations (class B), as shown in Table S3 and Figure S3. On average, the orientation changed to the intermediate class A/B for both mt2C9 and mt2C19 (see Table 1). The membrane insertion depths of the globular domain and F'–G' region of the chimeras showed small shifts towards the depths of the other protein.

#### *2.5. All-Atom MD Simulations Based on the CG Simulations Result in Stable Systems for Both Isoforms.*

We next performed all-atom (AA) MD simulations to obtain refined atomic-level interactions between the membrane and the proteins. Two different initial configurations obtained from the CG simulations (S1 and S2) were used for AA simulations of each isoform. In each case, two simulations (SIM1, SIM2) were performed starting with different initial velocities assigned from the CG:S1 configuration, one for the apo from and one for a ligand-bound form, and one simulation (SIM3) was performed starting from the CG:S2 configuration.

For both proteins, the structure of the globular domain was well preserved in these simulations, as indicated by the Cα-atom root mean squared deviation (RMSD) of the globular domain with respect to the initial energy minimized structure (see Figure S4). The structures stabilized with an RMSD of about 2–2.5 Å during the simulations. Furthermore, comparison of computed and crystallographic B-factors showed that the regions of the globular domain with large fluctuations were regions with high crystallographic B-factors (see Figure S5). This was the case for both isoforms, even though the crystallographic B-factors indicated somewhat different flexible regions in the crystal structures (see Figure S6). In CYP 2C19, the HI loop, the meander region (consisting β-bulge region and the K helix region), and β-strand 3 (residues 460–475) had higher B-factors (see Figures S5 and S6). The presence of the membrane did not restrict the flexibility of the membrane interacting regions, such as the linker preceding the A-helix, the B–C loop, and the F'–G' region, and strands β1-1, β1-2, β2-1, and β2-2. Indeed, partial unwinding of the G'-helix was observed in the simulations of CYP 2C19 (2C19:SIM1). The high linker flexibility contributed to higher fluctuations in the transmembrane helix angle, γ, in this particular simulation (see Table 2).

**Table 2.** Positioning of the CYP globular domain with respect to the membrane in the AA MD simulations of CYP 2C9 and CYP 2C19. Values of the mean and standard deviations of angles (as defined in Figure 2) and distances characterizing the positioning of the globular and TM domains were calculated for the last 50 ns of all-atom simulations, with different starting configurations from CG simulations and with different initial velocities assigned. The orientations of the globular domain were assigned to class A, A/B, or B.


<sup>1</sup> Simulated with a ligand in the active site (all other simulations were for the apoproteins). For CYP 2C9, the ligand was flurbiprofen from the crystal structure, and for CYP 2C19, it was the inhibitor from the crystal structure (Protein Data Bank chemical component 0XV). <sup>2</sup> CG simulations were run for an average of 10 μs. The angles were computed for the representative structure that was selected for starting AA MD simulations.
