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The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI)
has been determined to delineate the structural basis for the observed
differences in binding affinity and thermodynamics relative to
4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI
complex, there is a shift in the 1-CPI complex of the protein backbone in
helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301,
and leading to significant reshaping of the active site. Phe-206 and Phe-297
exchange positions, with Phe-206 becoming a ligand-contact residue, while
Glu-301, rather than hydrogen bonding to the ligand, flips away from the active
site and interacts with His-172. As a result the active site volume expands from
200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two
structures, it was predicted that a Phe-206-->Ala substitution would alter
1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments
indicated that this substitution had no effect on the thermodynamic signature of
4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A
showed significantly less favorable entropy but more favorable enthalpy. This
result is consistent with loss of the aromatic side chain and possible ordering
of water molecules, now able to interact with Glu-301 and exposed residues in
the I-helix. Hence, thermodynamic measurements support the active site
rearrangement observed in the crystal structure of the 1-CPI complex and
illustrate the malleability of the active site with the fine-tuning of residue
orientations and thermodynamic signatures.
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