Flexibility of Human Cytochromes P450: Molecular Dynamics Reveals Differences between CYPs 3A4, 2C9, and 2A6, which Correlate with Their Substrate Preferences

Abstract

Molecular dynamics (MD) simulations at normal and high temperature were used to study the flexibility and malleability of three microsomal cytochromes P450 (CYPs): CYP3A4, CYP2C9, and CYP2A6. Comparison of B-factors (describing the atomic fluctuations) between X-ray and MD data shows that the X-ray B-factors are significantly lower in the regions where the crystal contacts occur than for other regions. Consequently, the conclusions about CYP flexibility based solely on the X-ray data might be misleading. Comparison of flexibility patterns of the three CYPs enabled common features and variations in flexibility and malleability of the studied CYPs to be identified. The previously described pattern of flexibility in topological elements of microsomal CYPs (a rigid heme binding core, a malleable distal side and intermediately flexible proximal side) was confirmed. These topological features provide an important combination of high stereo- and regio-specificity (mediated by the relative rigidity in the neighborhood of the heme), together with high substrate promiscuity due to the more flexible active site and the malleability of the distal side. The data acquired here show that the malleability of the three studied CYPs correlates with their substrate specificity: CYP2A6 has a narrow substrate range and is the most rigid, CYP3A4 is the most promiscuous CYP known and is the most malleable, and CYP2C9 is intermediate in terms of both its substrate specificity and malleability. Thus, the malleability of CYPs is probably a major determinant of their substrate specificity.

Effect of Conformational Dynamics on Substrate Recognition and Specificity as Probed by the Introduction of a de Novo Disulfide Bond into Cytochrome P450 2B1

“…The flexible regions include the F-and G-helices, B/BЈ, D/E, F/G, J/JЈ, KЈ/KЉ, and ␤ 3-1 /␤ 3-2 loops. The flexible regions observed in the MD simulations are consistent with the plastic regions identified by Halpert's group (6) and resemble the flexible regions of CYP2C9 identified previously by MD simulations (8). Our MD simulations also show that the conserved regions in the vicinity of the heme such as the I-and K-helices and ␤-bulge are less mobile.…”

“…These authors proposed that the extreme flexibility of CYP3A4 accounts for its promiscuity, as CYP3A4 metabolizes nearly ϳ50% of all clinically used drugs. The complexity of the conformational flexibility and dynamics are also revealed in an MD simulation study of CYP3A4, 2C9 and 2A6 (8). Importantly, this molecular dynamics (MD) simulation study shows that the three-dimensional structure of P450s is more flexible in solution than was observed in the crystal structure.…”

“…Channel 2 connects the active site void to the exterior around the B/BЈ-C loop. This seems to be one of the more common access channels, as it has been found in several bacterial and mammalian P450s (8,33,39). Channel 3a passes between the E-, F-, and I-helices and ␤ 3-2 .…”

“…Channel 3a is situated on top of the heme on the distal side and most accessible to the active site. This channel is the preferred access channel for CYP2C9 and the only channel seen in CYP2D6 based on two independent MD simulations (8,33). Based on a recent x-ray crystallographic study of the three-dimensional structure of human aromatase, Ghosh et al (32) proposed that this channel is the main transport route for steroids and most likely is in close contact with lipid membranes for transporting lipophilic substrates like steroids.…”

“…This access channel has been proposed to be the putative access channel for androgen in human aromatase (CYP19A1) based on a crystallographic study of androstenedione-bound CYP19A1 (32). It also resembles the solvent channels found in CYP102A1, CYP2D6, and 2C5 that provide conduits for rapid access to the active site from the protein surface (8,33,40).…”