TY - JOUR
T1 - Allosteric Interactions in Human Cytochrome P450 CYP3A4
T2 - The Role of Phenylalanine 213
AU - Denisov, Ilia G.
AU - Grinkova, Yelena V.
AU - Nandigrami, Prithviraj
AU - Shekhar, Mrinal
AU - Tajkhorshid, Emad
AU - Sligar, Stephen G.
N1 - Funding Information:
The authors acknowledge the contribution of A. McInerney (University of Illinois at Urbana-Champaign) to expression and experimental studies of F213 mutants in the early stage of this work. This work made use of the Illinois Campus Cluster, a computing resource that is operated by the Illinois Campus Cluster Program (ICCP) in conjunction with the National Center for Supercomputing Applications (NCSA) supported by funds from the University of Illinois at Urbana-Champaign. All simulations have been performed using XSEDE resources (Grant MCA06N060).
Funding Information:
*E-mail: s-sligar@illinois.edu. ORCID Emad Tajkhorshid: 0000-0001-8434-1010 Stephen G. Sligar: 0000-0002-5548-2866 Funding This work was supported by National Institutes of Health MIRA Grant R01-GM118145 to S.G.S. and Grants R01-GM101048 and P41-GM104601 to E.T. Notes The authors declare no competing financial interest.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/3/12
Y1 - 2019/3/12
N2 - The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.
AB - The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.
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U2 - 10.1021/acs.biochem.8b01268
DO - 10.1021/acs.biochem.8b01268
M3 - Article
C2 - 30785734
AN - SCOPUS:85062513381
SN - 0006-2960
VL - 58
SP - 1411
EP - 1422
JO - Biochemistry
JF - Biochemistry
IS - 10
ER -