*2.7. Key Residues Contribute to Di*ff*erences in the Membrane-Protein Interactions of CYP 2C9 and CYP 2C19*

From the CG and AA MD simulations, we found that despite high sequence conservation (92% sequence identity), the two isoforms of the CYP2C subfamily, CYP 2C9 and CYP 2C19, maintained differences in the interactions, orientations, and degree of insertion in the membrane of the globular domain. The most important differences in residues were found in the substrate recognition sites (SRSs). For example, the SRS1- a, SRS1- b, SRS1 and SRS2,3 regions defined by Zawaira et al. [24] covered residue differences in the linker region (G46D), between strands β1-1 and β1-2 (72–73 KP-ER), in the B–C loop (I99H) and between the F' and G' helices (220–221 SP-PT) (see Figure 1).

We therefore analyzed the trajectories to differentiate residues interacting with the membrane head or tail regions, calculated by defining a 5 Å distance cutoff between protein and lipid head group (phosphate atoms) and hydrophobic tail, separately. The % contact time or occupancy of these residues with the membrane components in the trajectories is shown in Figure 5A. In the simulations, the interactions of the globular domains of both isoforms with the membrane were mainly developed through strands β1-1 and β1-2 (residues 64–74), and the F'–G' region (residues 210–226) (see Figure 5A,B). However, CYP 2C9 showed further interactions with the membrane through the A-helix (residues 50–60), the B–C loop (residues 95–110) and the C-terminal β-sheet 2 (residues 370–385). The peripheral interactions developed by CYP 2C9 were similar to the hydrophobic surface identified in CYP 2C5 (residues 30–45, 60–69 after the A-helix, 376–379 in β-strand 2-1 and the F'–G' helices) [28]. These secondary interactions, with either lipid tail or head regions, were established by the SRS regions that showed primary sequence differences in the two CYP isoforms studied here. Therefore, differences in the above-mentioned SRS regions can be crucial for CYP-membrane interactions and the orientation of the globular domain, and may lead to distinct substrate specificity.

We extended our sequence comparison to include the four main human CYP2C family members, CYP 2C8, 2C9, 2C18, and 2C19, and rabbit CYP 2C5 to examine the residues differing at the interface region (see Figure 5**C**). Sequence comparison showed that only CYP 2C19 had a positively charged residue, R73, at this position, as all other CYP2C members had P73. Furthermore, only CYP 2C9 had a positively charged residue at position 72 (K72) when compared to the other CYPs; this residue could play a role as a selectivity filter for attracting the acidic substrates preferred by CYP 2C9 and repelling basic compounds. In the F'–G' region, the S220 and P221 residues were only found in CYP 2C9, whereas the polar residue T221 was present only in CYP 2C19. Substitution of S220P and P221A in the CYP 2C9m7 structure (PDB 1OG2) shifted the position of P221 to that of P220, as in all other CYP2C members, which resulted in a turn between the F'–G' helices and further stabilized the G'-helix. Experimentally, it has been observed that the substitution of CYP 2C9 residues I99H, S220P, and P221T enhanced omeprazole 5- -hydroxylation activity of CYP 2C9 [18]. In another experimental study, E72K substitution in CYP 2C19 decreased the metabolic activity, whereas the K72E substitution in CYP 2C9 increased the binding affinity of tricyclic antidepressant (TCA) drugs such as imipramine [21]. Together, the analysis of primary sequence, protein-membrane orientation and interactions, and experimental findings, supports the role of different residues in SRS regions in determining the distinct orientations of the two CYPs, leading to differences in substrate access and selectivity.

**Figure 5.** Differences in the arrangements of the CYP 2C9 and CYP 2C19 residues at the membrane interface. (**A**) Residues in contact with the lipid head group (blue) and tail region (red) during the AA MD simulations of CYP 2C9 (above) and CYP 2C19 (below). The percentage of snapshots in which a contact was present is shown on the y-axis, and the residues interacting with the membrane are given on the x-axis. The secondary structures and substrate recognition sites are shown on the top. The residues differing in the interacting regions between CYP 2C9 and CYP 2C19 are labeled. (**B**) For CYP 2C9 (left) and CYP 2C19 (right), the last frames from AA MD simulations of the apo form (SIM1) are shown for the full system and for the membrane interface region. The protein is shown in cartoon representation with selected side chains in ball-and-stick representation colored by atom type with cyan carbons. The linker is shown in orange, β-sheets 1 and 2 in magenta, the B–C loop in yellow, the F and G helices in red, the F' and G' helices in green, the central I-helix in blue, and the heme and key residues in cyan licorice representation. The POPC bilayer is shown in grey line representation with phosphate atoms as red spheres. The magenta arrows indicate differences in β-sheet 2 (residues 370–380). (**C**) Part of a sequence alignment of human CYP2C subfamily members (2C9, 2C19, 2C18, 2C8) and rabbit CYP 2C5. The conserved residues are shown in red for similar residues or with a red background and in white for identical residues. The residues outside the blue boxes differed amongst the aligned CYPs.

#### *2.8. Structural Di*ff*erences Result in Di*ff*erent Membrane-Protein Interactions*

Structural analysis of CYP 2C9 and CYP 2C19 (PDB 1R90 and 4GQS, respectively) revealed different conformations of β-sheets 1 and 2 and the B–C loop (highlighted by rings in Figure 5B and which were observed in simulations of apo and ligand-bound proteins (Figure S7). During simulations, strands β1-1 and β1-2 remained inserted in the membrane or interacted with lipid head groups in CYP 2C9, whereas these strands made far fewer contacts in CYP 2C19. The conformation and orientation of strands β1-1 and β1-2 favored the interaction of β-sheet 2 (residues 370–385) with the membrane head groups in CYP 2C9, whereas this interaction was almost completely absent in CYP2 C19 (Figure 5A).

β-sheet 1 in CYP 2C9 differed in sequence (residues K72–P73) from CYP 2C19, (residues E72–R73), as well as in conformation (see Figure 5C). The turn between strands β1-1 and β1-2 in CYP 2C9 pointed away from the globular domain towards the membrane surface (see Figure 5B). K72 in CYP 2C9 pointed towards the binding pocket and, during the simulations, its positively charged ε-amino group transiently formed a hydrogen bond with S220 in the F'–G' helices and with the phosphate head groups of lipid molecules (see Figure 6C). The interaction with the lipid head groups resulted in the further insertion of the β1-hairpin residues into the membrane (Figure 6). K72 has been suggested to play an important role in the selection of anionic substrates in CYP 2C9, and is positioned along pathway 2b (Figure 6, a description of ligand pathways is given by Cojocaru et al. [29]) for ligand entrance into the binding pocket from the membrane [21]; it is replaced by E72 in CYP 2C19. Besides K72, the presence of P73 in CYP 2C9 favored interactions with the hydrophobic interior of the membrane (also seen in the % occupancy contact plot, Figure 5A). Thus, the K72 and P73 residues of the β-hairpin (between the β1-1 and β1-2 strands) in CYP 2C9 could be determinants of the difference in the orientation of the globular domain in the membrane compared to CYP 2C19.

In the simulations of CYP 2C19, the β1-1 and β1-2 strands showed fewer interactions with the membrane surface, which could be attributed partly to its charged residues E72 and R73 and the conformational differences observed in the crystal structure. In various studies on membrane–protein interactions, it has been found that arginine has a propensity to stay in the lipid head group region [30–33]. In CYP 2C19, the R73 sidechain pointed towards the membrane and, thereby, appeared to restrict insertion of the β-strands in the membrane. Together, the differences in the interactions with the membrane resulted in greater tilting of the distal side of the globular domain towards the membrane, resulting in higher β angles and higher heme-tilt angles, for CYP 2C19 than for CYP 2C9 (see Figure 5B).

An important difference between CYP 2C9 and CYP 2C19 was seen in the B–C loop, which differed in only one residue, residue 99 (I in CYP 2C9, H99 in CYP 2C19). The B–C loop in CYP 2C9 was highly mobile compared to CYP 2C19, in which a B'–C' helical conformation was present. The B–C loop also differed in the side chain conformations of R105 and R108 in the two CYPs. R105 in CYP 2C19 pointed outward and showed electrostatic interactions with D224 in the G' helix. In the CYP 2C9 crystal structure (PDB 1R9O), R105 had a different conformation and no interactions were reported with D224 due to the missing G'-helix. However, after modeling of the F'–G' helices and simulations, similar interactions between R105 and D224 were observed in most simulations.

From the CG simulations, we identified a role for the F'–G' helices in stabilizing the interactions and orientation of the globular domain in the membrane. The AA simulations showed differences between the two CYPs in the F'–G' helices (S220P and P221T). P221 in CYP 2C9 was located on the outer surface of the G'-helix, which is in direct contact with the membrane and thereby favored insertion of P221 into the lipid tail region, whereas T221 at the same position in CYP 2C19 made slightly less contact with the bilayer.

Together, these results imply that not only sequence differences but also conformational differences in the regions involved in membrane-protein interactions contribute to the differences in the orientations adopted by the two isoforms in the membrane.

**Figure 6.** Initial (**A**) and final (**B**) snapshots of the AA MD simulation (SIM1) of the apo form of CYP 2C9, showing tunnels accessible to a water molecule probe between the active site and the protein surface. Tunnel 2b (green) connects the active site and the membrane and is present in both snapshots, as is tunnel S (cyan). Tunnel 3 (yellow) is present in the initial snapshot and tunnel 2c (blue) is present in the final snapshot. The protein and bilayer are shown with the same color scheme as in Figure 5. (**C**) Close-up view of the entrance to the 2b tunnel showing how the phosphate group of a phospholipid molecule (shown in stick representation colored by atom type with cyan carbons) makes a hydrogen bond with the amino group of K72 (all other lipid molecules are represented by spheres for the phorphorous atoms only; the protein is represented and colored as in Figure 5). This interaction is important for pulling the phospholipid molecule somewhat out of the membrane towards the tunnel to the active site. This motion leads to partial opening of the β-sheet and the F'–G' regions; further opening would be required for a substrate molecule to access the active site. (**D**) K72 may interact analogously with acidic substrates, such as flurbiprofen, a drug that is a substrate of CYP 2C9 (left), and may repel basic substrates such as the tricyclic antidepressant (TCA) drug, imipramine, which is a substrate of CYP 2C19 (right).
