Dynamics and Hydration of the Active Sites of Mammalian Cytochromes P450 Probed by Molecular Dynamics Simulations

Abstract

The flexibility, active site volume, solvation, and access path dynamics of six metabolically active mammalian cytochromes P450 (human 2A6, 2C9, 2D6, 2E1, 3A4 and rabbit 2B4) are extensively studied using molecular dynamics (MD) simulations. On average, the enzymes' overall structures equilibrate on a 50+ ns timescale. The very open CYP2B4 structure closes slowly over the course of the simulation. The volumes of the active sites fluctuate by more than 50% during the MD runs; these fluctuations are mainly due to movements of the main chains, with only a handful of amino acid residues in CYP2B4, CYP2D6, CYP2A6 and CYP2C9 showing significant independent side chain movement. The volume of the active site of CYP2E1 fluctuates heavily, ranging from 220 to 1310 A(3), due to the opening and closing of gates to two adjacent cavities. CYP2E1 has the least hydrated active site of the studied CYPs; this is consistent with its preference for non-polar substrates. The CYP2A6 and CYP2E1 active sites are deeply buried, with access paths that are narrower than the radius of a water molecule. However, waters are still able to access these active sites due to local adaptations of the channel to accommodate their passage. This finding may imply that the access paths of the CYPs never fully open prior to contact with the substrate; instead, the substrate may induce adaptive conformational changes during its passage to the active site. This may also explain why some substrate recognition sites are localized along individual enzymes' access paths.

Anatomy of enzyme channels

“…The frequencies of amino acids in the middle regions of the channels correspond to the frequencies in the entire channel with the exception of glycine (Gly) and aromatic amino acids (Trp, Tyr, Phe), which are present more frequently. We may hypothesize that the higher frequency of glycine (Gly) in the middle channel parts is because it facilitates flexibility, which may be important for substrate/product channeling between the active site and protein surface [29], whereas aromatic amino acids can serve as gate-keepers. External parts of the channel bear more charged residues than any other part (Arg, Lys, Glu, Asp) together with proline (Pro) and glutamine (Gln), whereas other polar residues (Ser, Thr) are surprisingly less common in the external parts even though that both Ser and Thr are evenly distributed within the structure of proteins (Additional file 1: Figure S4).…”

“…The amino acids lining the access channels of cytochrome P450 (CYP) are important for the selectivity of these enzymes [28] while the flexibility of these channels, i.e. their opening and closing motions, contributes to the broad substrate specificity of CYP [10,29]. …”

Structural basis for cooperative binding of azoles to CYP2E1 as interpreted through guided molecular dynamics simulations

“…First, active site water molecules were absent in the X-ray crystal structures of CYP2E1 with either 4-methylpyrazole or indazole [26], which was reasonable given the well-characterized hydrophobicity of the CYP2E1 access channel and active site. Second, previous MD simulations on the hydration of P450 active sites demonstrated that the access channel for CYP2E1 was often less than the diameter of a water molecule [27]. This restriction on movement of water and inherent hydrophobicity of the CYP2E1 active site and access channel would minimize the possibility of hydration within the enzyme.…”

Interaction of selected platinum(II) complexes containing roscovitine-based CDK inhibitors as ligands with human liver microsomal cytochrome P450

“…macrolide antibiotics and steroids) (ref. [34][35][36][37] ). We reported earlier 38 that oxaliplatin exhibits only negligible effect on P450s even at very high concentration.…”