RT Journal Article
SR Electronic
T1 CYP2C8 Exists as a Dimer in Natural Membranes
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1976
OP 1983
DO 10.1124/dmd.110.034942
VO 38
IS 11
A1 Hu, Gang
A1 Johnson, Eric F.
A1 Kemper, Byron
YR 2010
UL http://dmd.aspetjournals.org/content/38/11/1976.abstract
AB CYP2C8 with a modified N-terminal sequence (2C8H) crystallizes as a dimer, but it is not known whether native CYP2C8 exists as a dimer in natural membranes. We have examined the organization of 2C8H and CYP2C8 expressed in bacterial membranes and mammalian endoplasmic reticulum membranes, respectively, by cysteine scanning and cross-linking or oxidation of sulfhydryl groups. In both forms of CYP2C8, cross-linked dimers were observed that were eliminated by mutation of Cys-24 in the linker region. Introduction of individual cysteines in the N-terminal 21-amino acid membrane-spanning signal anchor resulted in a pattern of cross-linking consistent with an α-helical structure for the signal anchor. In the linker region, cross-linking was observed for cysteine substituted at residues 22, 23, or 24, just before three Arg residues, indicating close apposition of the two linker sequences despite the neighboring positive charges. Introduction into the F-G loop region of cysteine pairs optimally located for cross-linking based on the crystal structure resulted in cross-linked dimers in the Cys-24 mutant. Deletion of the signal anchor sequence eliminated cross-linking mediated by Cys-24 or by cysteines introduced in the F-G loop regions, indicating that the signal anchor interaction is required for stable dimer formation. These results indicate that the signal anchor sequence and the F-G loop region form interfaces for CYP2C8 intermolecular interactions in natural membranes.