Abstract
To probe the possible substrate exit channel(s) in cytochrome P450 (P450) 2B1 and to clarify the role of residues previously identified by site-directed mutagenesis, a homology model was constructed based on the X-ray crystal structure of a P450 2B4-inhibitor complex. Testosterone was docked into the active site of P450 2B1 and was then pulled out through three putative channels using steered molecular dynamics simulations. The results indicated that of the three channels, the “solvent channel,” lined by helices E, F, and I and the β3 hairpin, required the largest rupture force and backbone motion, which rendered it unlikely as an exit route. The relatively small rupture forces and backbone motions for the other two channels suggested them as possible candidates for testosterone passage. The opening of channel 1, located between helices G and I and the B′-C loop, is characterized by rotation of the aromatic ring of Phe297 together with a bending of the B′-C loop. The opening of channel 2, penetrating through the B′-C loop/B′ helix, is achieved by an expansion of this region and a small displacement of the backbone. Interestingly, during the egress of testosterone along channel 1, Phe297 and Phe108 appear to act as two clamps to stabilize testosterone binding and prevent it from leaving the active site. Phe115 acts as a gatekeeper for channel 2. These results are in agreement with previous site-directed mutagenesis experiments.
Footnotes
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Financial support from the following institutions is gratefully acknowledged: State Key Program of Basic Research of China (Grant 2002CB512802), the National Natural Science Foundation of China (Grants 20372069, 29725203, 20472094, and 20102007), the Basic Research Project for Talent Research Group from the Shanghai Science and Technology Commission, the Key Project from the Shanghai Science and Technology Commission (Grant 02DJ14006), the Key Project for New Drug Research from the Chinese Academy of Science, the Qi Ming Xing Foundation of Shanghai Ministry of Science and Technology (Grant 03QD14065), and the 863 Hi-Tech Programme (Grants 2002AA233061, 2002AA104270, 2002AA233011, and 2003AA235030). These studies were also supported by Grant ES03619 and Center Grant ES06676 from the National Institutes of Health of the United States.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.004200.
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ABBREVIATIONS: P450, cytochrome P450; SMD, steered molecular dynamics; 3D, three-dimensional; PDB, Protein Data Bank; MD, molecular dynamics; RMS, root-mean-square; RMSD, RMS deviation; RMSF, RMS fluctuation.
- Received February 11, 2005.
- Accepted April 6, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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