*2.1. CG Simulations Show That the CYP 2C9 and CYP 2C19 Globular Domains Adopt Distinct Orientations in the Membrane Bilayer*

The coarse-grained (CG) simulations carried out are listed in Table S1. The trajectories were analyzed to assess convergence of the orientation of the globular domain and its interactions with the membrane. Different starting structures, different lengths of the flexible linker region, and different simulation parameters were tested to ensure that reliable CYP-membrane interactions and globular domain orientations were obtained. The converged positions and orientations from all sets of simulations of CG systems are given in Table 1.

Simulations of the full wild-type sequences of the two isoforms (systems S1 and S2) show different orientations from each other in the membrane. For the full-length wild-type CYP 2C9, the angles α and β (see Figure 2 and Materials and Methods for definition of these angles) ranged from 89 to 91◦ and 112 to 118◦, respectively. For the full-length wild-type CYP 2C19, the α and β angles ranged from 97 to 100◦ and 134 to 137◦, respectively. The axial distance of the center of mass (CoM) of the globular domain from the membrane CoM was 42–43 ± 2 Å for CYP 2C9 and 47–48 ± 2 Å for CYP 2C19. The different orientations of the two isoforms were classified into three different classes, A, A/B, and B. When the β angle was below 125◦, the orientation was categorized in class A, from 125◦ to 130◦ into class A/B, and above 130◦ into class B. All wild-type CYP 2C9 CG systems (S1–S3) converged to class A, and all wild-type CYP 2C19 CG systems (S1–S3) converged to class B. The angles and distance values were plotted for all the CG systems in Figure S1. Thus, the two isoforms adopt distinct positions and orientations of their globular domains in the phospholipid bilayer in the CG simulations.

**Table 1.** Results of CG simulations of CYP 2C9 and CYP 2C19 for the full length (S1, S2) and truncated (S3) wild-type proteins (based on crystal structures 1R9O and 4GQS, respectively), various models of full length CYP 2C9 (M1–M4, based on structures Model 1–4), and the mutant chimeras, mt2C9 and mt2C19. The systems simulated are listed in Table S1. The mean and standard deviation values are given for parameters defining the position of the globular domain with respect to the membrane: angles α and β (defining the orientation, as shown in Figure 2 and class (A, A/B, or B)) and the axial distances of the center of mass (CoM) of the bilayer from the CoM of the linker, the F'–G' region, and the globular domain, respectively. The simulations each had an average duration of 10 μs, and the parameters were computed for snapshots from the last 9 μs collected at intervals of 1 ns.


<sup>1</sup> Systems containing only the CYP globular domain, residues 47–490 for both proteins, without the TM and linker regions.
