Plasticity of Cytochrome P450 2B4 as Investigated by Hydrogen-Deuterium Exchange Mass Spectrometry and X-ray Crystallography

Abstract

Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B-and C-helices and portions of the F-Ghelix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior. Cytochrome P450 (P450)4 dependent monooxygenases are involved in the biogenesis of sterols and hormones and oxidation of a broad range of xenobiotic compounds (1). Many individual mammalian cytochromes P450 can accept a wide variety of hydrophobic substrates of differing shapes and sizes and render them more hydrophilic for excretion or subsequent conjugation. In addition to their central role in drug clearance, the ability of mammalian cytochromes P450 to convert various inactive precursors to the respective bioactive compounds makes these enzymes of paramount importance for the healthcare and pharmaceutical industries (2-4).Despite their broad range of substrates, the single domain fold of P450s is well conserved across families (5-9). Hence, the ability to adapt to molecules reflects the notable plasticity of many secondary structural elements (8, 9). P450s show the ability to form compact structures around small ligands or empty active sites, as evidenced by P450 2B4 complexed with 1-(4-chlorophenyl)imidazole (1-CPI) or 4-CPI (10, 11), P450 3A4 (12), and several proteins in the 2C subfamily (13,14). Moreover, some P450s also appear able to alter their conformations to accommodate ligands of greater volume, as seen in P450 2B4 with bifonazole (15) or in P450 3A4 with erythromycin or with ketoconazole (16).Our laboratory has utilized an engineered form of P450 2B4 termed P450 2B4dH 5 (N-terminally modified and a C-terminal His tag) to gain insight into enzyme flexibility (10,11,15,(17)(18)(19). The i...

Structures of Cytochrome P450 2B6 Bound to 4-Benzylpyridine and 4-(4-Nitrobenzyl)pyridine: Insight into Inhibitor Binding and Rearrangement of Active Site Side Chains

“…These include two distinct ligand-free states of protein, open and closed, as well as other conformations observed in complex with various inhibitors and drugs (Gay et al, 2010a). The crystal structures of the open ligand free form and two inhibitor-bound complexes were in agreement with the conformational changes observed in solution in recent hydrogen-deuterium exchange mass spectrometry experiments (Wilderman et al, 2010). Furthermore, the flexible regions of 2B4 affected by ligand binding were consistent between the solution studies and X-ray crystal structures.…”

Structure and Function of Cytochromes P450 2B: From Mechanism-Based Inactivators to X-Ray Crystal Structures and Back

“…This new ligand-free structure (Fig. 1C) (Wilderman et al, 2010) differed significantly from the previously reported open ligand-free structure (Fig. 1B) (Scott et al, 2003), showing that CYP2B4 is able to adopt a closed conformation in the absence of ligand that closely resembles the 4-CPI and 1-CPI complexes.…”

“…1B) (Scott et al, 2003), showing that CYP2B4 is able to adopt a closed conformation in the absence of ligand that closely resembles the 4-CPI and 1-CPI complexes. Whereas the backbone maintains an RMSD of Ͻ0.8 Å compared with the other CYP2B4 closed structures, the active site cavity is intermediate to the smaller volume of the 4-CPI active site and the larger volume of the 1-CPI active site (Gay et al, 2010a;Wilderman et al, 2010). This change in volume is mostly due to the movement of Glu301 out of the active site (Fig.…”

Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins

“…Solubilization and crystallization of the former group of MPs also require the use of detergents to sequester hydrophobic protein patches and prevent protein aggregation. By mixing them with commercial detergents, FAs have been successfully used to crystallize several mammalian P450 enzymes [CYP24A1 (27), CYP2B4 (28)(29)(30)(31)(32), and CYP2B6 (32)(33)(34)]. Here, we provide a summary of these results (Table S4).…”