doi:10.1016/j.bbapap.2005.07.044
Copyright © 2005 Elsevier B.V. All rights reserved.
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Multiple molecular recognition mechanisms. Cytochrome P450—A case study
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Rebecca C. Wadea, 
, 
, , Domantas Motiejunasa, Karin Schleinkoferb, Sudarkoc, Peter J. Winna, Amit Banerjeed, Andrei Kariakine and Christiane Junge
aMolecular and Cellular Modeling Group, EML Research, Schloss-Wolfsbrunnenweg 33, 69118 Heidelberg, Germany
bDepartment of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
cDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, University of Jember, Jl. Kalimantan 37, Jember 68121, Indonesia
dDepartment of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, USA
eProtein Dynamics Laboratory, Max Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany
Received 11 July 2005;
revised 18 July 2005;
accepted 19 July 2005.
Available online 16 September 2005.
Abstract
Biomolecular recognition is complex. The balance between the different molecular properties that contribute to molecular recognition, such as shape, electrostatics, dynamics and entropy, varies from case to case. This, along with the extent of experimental characterization, influences the choice of appropriate computational approaches to study biomolecular interactions. Here, we present computational studies of cytochrome P450 enzymes and their interactions with small molecules and with other proteins. These interactions exemplify some of the diversity of molecular determinants of binding affinity and specificity observed for proteins and we discuss some of the challenges that they pose for molecular modelling and simulation.
Keywords: Molecular recognition; Cytochrome P450; Molecular dynamics simulation; Protein dynamics; Protein docking
Abbreviations: FTIR, Fourier transform infra-red spectroscopy; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; P450, cytochrome P450; Pdx, putidaredoxin; RAMD, random acceleration molecular dynamics
Fig. 1. (A) Ribbon trace of a model of a mammalian cytochrome P450 attached to a phospholipid bilayer through its N-terminal helix and interactions of the F–G loop region. The heme cofactor is shown in CPK representation sequestered in the protein. (B) Schematic diagram showing intermolecular interactions of the P450 fold (cyan) with cofactors (light pink), cytochrome P450 reductase (CYP-R), lipid (green), substrate (S), product (P), as well as post-translational modification (yellow).
Fig. 2. Superposition of the experimental structures of putidaredoxin used for docking (X-ray: yellow; NMR: mauve). Differences are most apparent in the orientation of the C-terminal W106 and in the loop containing D34.
Fig. 3. Docked structures of CYP101 (green) and putidaredoxin obtained with two different structures of putidaredoxin (X-ray: yellow; NMR: mauve). Two major docking modes are obtained (shown left and right with respective detailed views of important interfacial residues and the cofactors below). These are similar for the two experimental structures in overall orientation but differ in the details of the interface residue interactions. The docked structures obtained with the X-ray structure of Pdx are consistent with available experimental data (see text for details).
Fig. 4. Egress routes identified in RAMD simulations of cytochrome P450cam (CYP101) [17]. The routes show representative trajectories of the centers of substrates or products egressing from the active site above the heme (shown in ball and stick representation). Routes are distinguished by numbers referring to the secondary structure elements lining them and letters referring to the spatial region where the ligand egresses. Pathway 2 a, lined by the B/C loop (with B' helix) (yellow), the F/G loop (blue), and the beta-1 hairpin (pink) is the dominant route found in three bacterial P450s studied [18]. Pathway 2 c, lined by the B/C loop, and the G and I helices, is the dominant route found in the mammalian P4502C5 [20].
Fig. 5. Superposition of 3 snapshots from a RAMD simulation of the egress of a substrate from the active site of the mammalian CYP2C5 via pw2c [20]. Selected helices are labeled. The initial bound conformation is shown in dark blue, an intermediate state in grey and a conformation after ligand expulsion in cyan. The side chains of K241 and V106 are shown. Ligand egress via pw2c requires the hydrogen-bond (shown by red dashed lines) between the K241 sidechain and the backbone oxygen of V106 to be broken.
Table 1.
Comparison of the characteristics of the dominant ligand egress pathway identified by RAMD simulations in three bacterial P450s (CAM, BM-3 and eryF) and one mammalian P450 (2C5)
1Max. Cα RMSF observed for a residue during the last 10-20 ps of ligand expulsion.
Corresponding author. Tel.: +49 6221 533 247; fax: +49 6221 533 298.
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