Abstract
The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.
Highlights
Human cytochrome P450 (CYP) enzymes play important roles in the metabolism of drugs, steroids, fatty acids, and xenobiotics
Since mammalian CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal helix and secondary contacts from the catalytic domain, differences in the sequence and 3D structure in the membrane-interacting region in the catalytic domain can lead to different membrane-protein interactions
As it has been hypothesized that lipophilic substrates enter into the binding pockets of CYPs from the membrane core, determining the orientation of CYPs in the membrane can provide insights into differences in the opening of ligand entrance tunnels to the membrane, substrate specificity, and the mechanism of drug selectivity
Summary
Human cytochrome P450 (CYP) enzymes play important roles in the metabolism of drugs, steroids, fatty acids, and xenobiotics. The CG simulations of the four CYP 2C9 models (M1–M4) indicated that it is the primary sequence and the initial conformational differences in the linker, B–C loop, and F’–G’ helix regions that influence the final positioning of the CYP globular domain in the membrane. The simulations of CYP 2C9 showed some readjustment in the orientations from the starting configurations in which the β angle increased from about 112◦ to 120–127◦, corresponding to remaining in class A in two cases (for the apoprotein and for the holoprotein with a substrate, the drug flurbiprofen, bound in the active site) and transitioning to class A/B in one case (SIM3), in which the globular domain structure was slightly less stable (see Figures S4 and S5). DDiiffffeerreenncceess iinn tthhee aarrrraannggeemmeennttssooff tthhee CCYYPP 22CC99 aanndd CCYYPP 2CC1199 resiidduueess aatt the memmbbrraannee iinntteerrffaace. ((AA))RReessiidduueessiinnccoonnttaaccttwwiitthhtthheelliippiiddhheeaaddggrroouupp((bblluuee))aannddttaaiillrreeggiioonn((rreedd))dduurriinnggtthheeAAAA MMDD ssiimmuullaattiioonnssooffCCYYPP22CC99((aabboovvee))aannddCCYYPP22CC1199(b(beleoloww).)T. hTehepeprecrecnetnatgaegoefosfnsanpasphsohtostisniwn hwichhicah caocnotancttawctaws apsrepsreensteinstsihsoswhnowonn tohne yth-aexyis-,aaxnisd, athnedrethsiedrueessidinuteesraincttienrgacwtiinthg twheitmh ethmebmraenme bareangeivaerne ognivtehneoxn-atxhies.xT-ahxeiss.eTcohnedsaercyonstdraurcytusrterus catnudresuabnsdtrsautebsrtercaotgenrietciongnsiittieosnasrietesshaorwe nshoonwtnheontotph.eTtohpe. rTehseidrueessidduieffsedriinffgerinintghieninthteerianctteinragcrtienggiornesgiboentws beetnwCeYenP C2CY9Pa2nCd9CaYndP C2CY1P92aCre19laabreeleladb.e(Ble)dF. o(rBC) FYoPr 2CCY9P(l2eCft9) (alnefdt)CaYnPd 2CCY1P92(Cri1g9ht()r,igthhet)l,atshtefrlasmt efrsafmroems fAroAmMADA sMimDusliamtiuonlastioofntshoefatphoe faopromfo(SrmIM(1S)IMar1e) sahreowshnowfonr tfhore tfhuellfusyllssteymsteamndanfdorfothr ethme emmebmrbanraenienitnetrefarfcaecerergeigoino.nT. hTehepprorotetieninisissshhoowwnniinnccaarrttoooonn rreepresentation wwiitthhsseeleleccteteddsisdideechcahianisnisnibnablla-alln-adn-sdt-isctkicrkeprreepsreensteantitoantioconlocroeldorbeydabtoymattoympetwypitehwcyiathn ccyaarbnocnasr.bTohnes.lTinhkeerlinisksehroiws snhionwonraingoer,aβn-gseh,eβe-tsh1eaentsd12ainndm2aignemntag, ethnetaB,–thCeloBo–pCilnooypellionwy,etlhloewF,athned FGahnedlicGeshienlirceeds,itnhreeFd’,atnhde FG’’ahnedlicGe’shinelgicresenin, tghreeceenn, ttrhael Ic-ehnetlriaxlinI-hbeluliex, iannbdluthee, ahnemd ethaenhdekmeye arensdidkueeys rienscidyuanesliicnorciycaenrelipcroersiecnetraetiporne.seTnhteatPioOnP.CThbeilaPyOePr Cis bshiloawyenr iins sghreoywlninienrgepreryesleinteartieopnrewseitnhtapthioonspwhiatthe pahtoomspshaastreeadtospmhsearessr.eTdhsepmheargeesn. tTahaerrmowagseinntdaicaartreowdisffienredniccaetseindiβff-esrheenecte2s (irnesβi-dsuheeset3720(–r3e8si0d).u(eCs)3P7a0r–t 3o8f0a).s(eCq)uPenacret oafliagnsmeqeunetnocfehaulimgnanmCenYtPo2fChsuumbfaanmCilYyPm2Cemsbuebrfsam(2iCly9,m2Cem19b,e2rCs1(82,C29C, 82)Ca1n9d, 2rCab1b8i,t2CCY8P) a2nCd5.raTbhbeitcoCnYsePrv2eCd5.reTshideuceosnasreervsehdowrensiidnureesdafroer sihmowilanr irnesrideduefsororsiwmitilhara resdidbuaecskgorrouwnitdhaandreidn bwahckitgerfoournideanntidcainl rweshiditueefos.rTidheenrteicsaidl ureessidouetssi.dTehtehreebsildueuebsooxuestsdidiffeetrheedbalmueobnogxset sthdeifafelirgendeadmCoYnPgss.t the aligned CYPs
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