Architecture of a single membrane spanningcytochrome P450 suggests constraints that orient thecatalytic domain relative to a bilayerBrian C. Monka,b,1, Thomas M. Tomasiakc,1, Mikhail V. Keniyaa,b, Franziska U. Huschmanna,b,d, Joel D. A. Tyndalld,Joseph D. O’Connell IIIc, Richard D. Cannona,b, Jeffrey G. McDonalde, Andrew Rodriguezc, Janet S. Finer-Moorec,and Robert M. Stroudc,2

aSir John Walsh Research Institute, bDepartment of Oral Sciences, Faculty of Dentistry, and dSchool of Pharmacy, University of Otago, Dunedin 9054,New Zealand; cDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; and eDepartment of Molecular Genetics,University of Texas Southwestern Medical Center, Dallas, TX 75390

Contributed by Robert M. Stroud, January 10, 2014 (sent for review September 30, 2013)

Bitopic integral membrane proteins with a single transmembranehelix play diverse roles in catalysis, cell signaling, and morpho-genesis. Complete monospanning protein structures are needed toshow how interaction between the transmembrane helix andcatalytic domain might influence association with the membraneand function. We report crystal structures of full-length Saccharo-myces cerevisiae lanosterol 14α-demethylase, a membrane mono-spanning cytochrome P450 of the CYP51 family that catalyzes thefirst postcyclization step in ergosterol biosynthesis and is inhibitedby triazole drugs. The structures reveal a well-ordered N-terminalamphipathic helix preceding a putative transmembrane helix thatwould constrain the catalytic domain orientation to lie partly inthe lipid bilayer. The structures locate the substrate lanosterol,identify putative substrate and product channels, and reveal con-strained interactions with triazole antifungal drugs that are impor-tant for drug design and understanding drug resistance.

Membrane proteins that span the lipid bilayer once consti-tute around 50% of all integral membrane proteins (1).

Although monospanning membrane proteins carry out numerouskey biological functions, including environmental sensing, organelle-specific catalysis, and the regulation of cell morphology, only in-dividual domains or subdomains are currently represented in theProtein Data Bank, and structural information about interactionsbetween their transmembrane domains and extramembranouscomponents is lacking. Cytochrome P450 proteins are prominentenzymes with orthologs found in all kingdoms of life. In eukar-yotes, microsomal members of this major family of mixed-func-tion mono-oxygenases contain a single transmembrane helix andcan be grouped in two broad functional categories: biodefense,such as the first phase of xenobiotic detoxification, and coremetabolism including reactions in sterol biosynthesis and fattyacid oxidation (2).The lanosterol 14α-demethylases or CYP51 enzymes, probably

the most genetically ancient of the cytochrome P450 families,play a central role in cholesterol or ergosterol biosynthesis (3).CYP51s carry out three consecutive mono-oxygenase reactioncycles to remove the 14α-methyl group from lanosterol to yield 4,4-dimethyl-cholesta-8,14,24-trienol, a key precursor in cholesteroland ergosterol biosynthesis, releasing water and formic acid (3).Because of the key roles that CYP51s play in yeast, filamentousfungi, and some parasitic protozoa, these enzymes are thera-peutic targets for antimicrobial agents, including fluconazole(FLC), voriconazole (VCZ), and itraconazole (ITC) (4). Fungalinfections play an increasingly significant role in disease, impactingagriculture ecosystems and human health, especially in immuno-compromised individuals (5–7) for whom antifungal resistancecontinually poses a threat (8). In humans CYP51 is being tested asa target for cholesterol-lowering drugs (9) and in antiangiogeniccancer therapies (10). A limited set of cytochrome P450 isoforms

(1A2, 2C8, 2C9, 2C19, 2D6, and 3A4) metabolize about 75% ofthe drugs currently used in medicine (11).Membrane-bound cytochrome P450s catalyze reactions in-

volving many hydrophobic substrates, including lipophilic drugsand sterols that partition from the lipid bilayer into the activesite. Identification of enzyme orientation relative to the lipidbilayer therefore is critical for understanding substrate entry intoand egress from the heme-containing active site (12). For theeukaryotic cytochrome P450 enzymes that contain transmembranesegments, most X-ray structures have been obtained after re-moval of the N-terminal transmembrane domain to facilitateexpression and crystallization (13–18), as is the case with manyother monospanning membrane protein structures. ResolvedCYP51 structures include the soluble enzyme fromMycobacteriumtuberculosis (19) and N-terminal truncated enzymes from Homosapiens (20), Trypanosoma cruzi (21), Trypanosoma brucei (22),and Leishmania infantum (23). The available eukaryotic cyto-chrome P450 structures show the catalytic subunit has a conservedfold surrounding a central prosthetic heme group. A proposedsubstrate channel to the membrane as well as interactions with

Significance

The absence in the Protein Data Bank of full-length structuresof bitopic membrane proteins with one transmembrane helix,probably because of difficulties with ordered crystallization,has limited understanding of how single-transmembrane heli-ces orient enzymes and sensors at the bilayer surface. X-raycrystal structures of full-length yeast lanosterol 14α-demethy-lase, a cytochrome P450, show how a helix spanning a singletransmembrane may lead to constraints on the orientation ofthe putative substrate entry portal from within the bilayer. Thecrystal structures also locate the substrate lanosterol, identifyputative substrate and product channels, and reveal constrainedinteractions with triazole antifungal drugs that are important fordrug design and understanding the drug resistance associatedwith orthologs of the enzyme found in fungal pathogens.

Author contributions: B.C.M., T.M.T., and R.M.S. designed research; B.C.M., T.M.T., M.V.K.,F.U.H., J.D.A.T., J.D.O., R.D.C., J.G.M., A.R., and J.S.F.-M. performed research; B.C.M. andT.M.T. contributed new reagents/analytic tools; B.C.M., T.M.T., M.V.K., F.U.H., J.D.A.T., J.D.O.,R.D.C., J.G.M., J.S.F.-M., and R.M.S. analyzed data; and B.C.M., T.M.T., and R.M.S. wrotethe paper.

The authors declare no conflict of interest.

Data deposition: The structures reported in this paper have been deposited in the ProteinData Bank, www.pdb.org [PDB ID codes 4LXJ (ScErg11p-6xHis + lanosterol) and 4K0F(ScErg11p-6xHis + itraconazole)].

See Commentary on page 3659.1B.C.M. and T.M.T. contributed equally to this work.2To whom correspondence should be addressed. E-mail: stroud@msg.ucsf.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1324245111/-/DCSupplemental.

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several inhibitors, including ketoconazole, posaconazole, FLC,and substrate analogs such as 14α-methylenecyclopropyl-Δ7–24,25-dihydrolanosterol, have been visualized (19, 20, 22, 24).Although bacterial and mitochondrial orthologs of cytochrome

P450s and the M. tuberculosis CYP51 are soluble enzymes, bio-informatic analysis of many microsomal cytochrome P450s led tothe detection of a hydrophobic surface in the catalytic domain andprediction of an N-terminal monospanning transmembrane do-main that has been shown to be important for subcellular targetingand membrane insertion (25–28). Multiple in vitro approacheshave suggested that a transmembrane anchor is not always re-quired for the catalytic activity of some microsomal preparationsof cytochrome P450s (29, 30). For example,M. tuberculosis CYP51specifically converts detergent-solubilized lanosterol to its deme-thylated product (31). However, the in vivo role of the membraneanchor in catalysis by other CYP51s that use membrane-associatedlanosterol has yet to be established. Antibody-binding accessibilityassays showed that most of the surface of CYP2B4 (32) was sol-vent exposed with the exception of the putative transmembranehelix and the loop between the F and G helices. About 30% of thecatalytic domain of CYP11A1 was inaccessible to chemical mod-ification by fluorescent probes (33). The physical displacement ofphospholipids in Langmuir–Blodgett monolayers indicated a con-siderably greater area of protein insertion than could be accountedfor by a single N-terminal transmembrane helix (34). This findingwas supported by atomic force microscopy, which showed thecatalytic domain protrudes only 3.5 ± 1 nm above the membrane(35). A crystal structure of full-length aromatase at 2.9-Å resolu-tion has been modeled, but weak electron density meant that thestructures of 44 N-terminal and seven C-terminal residues couldnot be determined (36). Models that place the mouth of the sub-strate channel in direct contact with the hydrophobic environmentof the lipid bilayer required the catalytic domain helices A′ and A to

be imbedded and partially imbedded, respectively, in the lipid bi-layer (28, 37).

ResultsArchitecture of the Cytochrome P450 Saccharomyces cerevisiaeLanosterol 14α-Demethylase. We report X-ray crystal structuresof full-length Saccharomyces cerevisiae lanosterol 14α-demethy-lase (ScErg11p-6xHis) with the membrane domain structurallydiscernable and two ligands cocrystallized: the substrate lano-sterol and the triazole inhibitor ITC. Clear electron density forthe 50 N-terminal amino acids revealed two helices orientedat ∼60° to each other (Fig. 1 A and B). The N-terminal helix[membrane helix 1 (MH1), residues 6–23] is amphipathic andmakes extensive crystal contacts with symmetry-related mole-cules (Fig. 2 A and B). The distribution of hydrophobic andhydrophilic side chains suggests that the hydrophobic face maycontact the inner leaflet of the endoplasmic reticulum (ER)membrane (Fig. 1B). MH1 connects to TMH1 via a short proline-containing turn (residues 24–26), a slightly kinked helix 37.5 Ålong (residues 27–51) that is of sufficient length to traverse thelipid bilayer. The side chains of amino acids R30 and Q29, ad-jacent to the turn, form a C-terminal cap for helix MH1 and anN-terminal cap for TMH1, respectively.Seventeen of the 25 residues in TMH1 are hydrophobic, with

a high concentration in the N-terminal portion, as is consistent withimmersion of TMH1 in the lipid bilayer. Beyond the proline kink atP38, TMH1 makes extensive polar contacts with the catalytic do-main (Fig. 1C). The helix then consists of opposing hydrophilic andhydrophobic faces that interact with the catalytic domain and lipidbilayer, respectively. An ionic interaction between the guanidiniumgroup of R52 and the carboxylate of D54 enables a sharp turnbetween TMH1 and the first loop of the catalytic domain.Paired polar residues constrain the orientation between

TMH1 and the catalytic domain. The e-amide group of Q46,which hydrogen bonds with the main-chain carbonyl and amideof Y61, is absolutely conserved in the fungal CYP51 family. Thehydroxyl of Y61 also makes a hydrogen bond with the δ-amidegroup of N42. Water molecules bridge the e-amide group of Q46with the main-chain amide oxygen of L58 and the ζ-hydroxyl ofY49 with the hydroxyl of S92. Other polar contacts in this region

Fig. 1. Overall fold of ScErg11p and predicted orientation in the lipid bilayer.(A) Helices are colored from the N terminus to the C terminus with a gradientfrom blue to red. The heme is shown as yellow sticks; lanosterol in the active-site cavity is shown in orange. (B) The structure of the amphipathic helix (MH1)and the transmembrane helix (TMH1). Hydrophobic side chains are coloredblue, and polar residues colored magenta, with oxygen atoms colored red, andnitrogen atoms colored blue. Difference electron density (Fo–Fc) contoured to2σ is depicted as a green mesh. (C) Interaction of the transmembrane domainwith the catalytic domain. Ordered water molecules are depicted as redspheres. Hydrogen bonding interactions are shown as dashed lines; distancesare in Å. (D) Distribution of hydrophobic and charged residues in the F/G loop.

Fig. 2. Crystal packing of ScErg11p-6xHis. (A) ScErg11p-6xHis crystals showalternating layers of transmembrane (TM) segments and the soluble seg-ments (S). (B) Crystal packing of two symmetry-related copies of ScErg11p-6xHis with the amphipathic helix S6–H21. The amphipathic helix is coloredgreen, and the symmetrically related copies are colored salmon or blue. (C)Overlay of lanosterol-bound (gray) and ITC-bound (salmon) ScErg11p-6xHis.

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include the guanidinium group of R55 and the carbonyl of A399;the main-chain amide of K53 and the hydroxyl of S397; and themain-chain carbonyl of D54 and the amide of A399. These exten-sive contacts bury a surface area of 51.8 Å2 on TMH1 and appear toform a rigid architecture that is important for establishing the poseof the enzyme in the bilayer (Figs. 1A and 2 A and C). The energeticstabilization conferred by the extensive contacts between TMH1and the catalytic domain make it unlikely that the crystal contactsbetween the amphipathic MH1 (residues S6, E10, E13, and H21)and residues K270 and S267 of one symmetry-related molecule andG532 and E354 of the other (Fig. 2B) would have distorted theoverall structure of ScErg11p significantly. The ScErg11p-6xHisstructures show few significant differences between structures, withthe most extensive changes (0.36 Å rmsd on Cα) being in the F/Gloop (Fig. 2C) of the lanosterol and ITC costructures.

Inferred Orientation of ScErg11p Relative to the Membrane. To es-tablish the membrane orientation, ScErg11p-6xHis was insertedinto preformed lipid vesicles and labeled from the outside at freecarboxylates with glycinamide in the presence of N′N′-Dicyclo-hexylcarbdiimide (DCCD) at pH 6 (Fig. 3A) (38). Tandem massspectrometry of tryptic digests identified labeled residues only onthe catalytic domain, with no labeling on the N-terminal MH1(residues 1–26), as is consistent with topological labeling of othercytochrome P450s and with MH1 and the catalytic domain as-sociating with opposite sides of the bilayer.The orientation of ScErg11p also was explored using two

algorithms for membrane insertion: the all-atomistic positioningof proteins in the membrane (PPM) procedure (39) and theknowledge-based Ex3d procedure (40). Both calculated poseswere very similar to those inferred from our crystallographicanalysis, i.e., TMH1 residues 27–50 link the N-terminal amphi-pathic helix MH1 and the C-terminal catalytic domain that are

partially submerged in opposite leaflets of the lipid bilayer (Fig.1A). The OPM analysis, which calculated a partitioning freeenergy of −31.3 kcal/mol into the lipid bilayer, was the best fitwith our topological labeling, including the labeling of residueE249 on the F/G loop, and was used in this analysis (Fig. 3).The pose we infer for ScErg11p relative to the membrane

buries portions of the loop connecting helices F–F′ and G in thelipid bilayer (Figs. 1D, 2C, and 3). It is consistent with muta-genesis experiments showing this region is important for mem-brane binding (33, 41, 42), fluorescence quenching analysis (5),the predicted pose of aromatase (36), and computational simu-lations for human cytochrome P450s performed in the presenceof diolyeolphosphatidylcholine (DOPC) (37) or 1-palmitoyl-2-oleoyl-sn-glycerol-2-phosphocholine (POPC) (27, 32–34, 37, 43).Several aspects of the structure support the proposed orientation.Hydrophobic residues are distributed in a single band across thesurface of the structure. Poisson–Boltzmann electrostatics calcu-lations using Delphi software (44) reveal an electrostatic potentialsurface showing a distinct hydrophobic/hydrophilic boundary (Fig.3B). Ordered water molecules are present only in proposed sol-vent-accessible areas of the model and are absent from the bilayer-accessible surface of the structure (Fig. 1 B and C). As a corollaryto the positioning of the lipid bilayer, the protein crystallized withalternating layers of proposed membrane and soluble segments(Fig. 2A) corresponding to the placement of hydrophobic andhydrophilic elements in the crystal structure, including the surface-associated ligands.We hypothesize that a cluster of charged residues found at the

N-terminal end of the G helix forms a major membrane-bindingcontact with the negatively charged phosphate head groups ofthe lipid bilayer (Fig. 1D). This orientation places the end ofa substrate channel in direct contact with the cytoplasmic surfaceof the lipid bilayer (Figs. 1A, 2C, 3B, and 4C). This substratechannel is bifurcated near its mouth, yielding a secondary ves-tibule (Fig. 3C) not previously identified in CYP51s. Although anN-decyl-β-D-maltoside (DM) detergent micelle differs from anintact lipid bilayer, the proposed orientation supports a model inwhich the substrate first is abstracted directly from the surface ofthe membrane via the juxtaposed channel mouth. After hemereduction and complex formation with oxygen, an oxygen re-bound mechanism converts the C14 methyl to the first of threeproducts that result in 4,4-dimethyl-cholesta-8,14,24-trienol (2).

Substrate and Product Binding. The enzyme was purified andcocrystallized with the substrate lanosterol or with four otherligands (itraconazole, estriol, FLC, or VCZ). The lanosterol (Fig.4A) and estriol (Fig. S1A) cocrystal structures showed densityconsistent with the common four-ring sterol in the active-sitecavity. A mixed state probably involving both endogenous lano-sterol and added ligand complicated interpretation of electrondensity in the active site. This mixed state did not occur whenScErg11p was purified in the presence of ITC and crystallized.As a result, only the lanosterol and ITC structures are detailed herewith the four others reported as models in SI Discussion.The lanosterol (Fig. 4A) and estriol (Fig. S1A) cocrystal

structures showed density consistent with the common four-ringsterol in the active-site cavity. Electron density consistent withthe tail of lanosterol is located in the primary vestibule that leadsup to the active site (Fig. 5B). Similar electron density was ob-served in the same locations when no ligand was supplied (Fig.S1D), indicating that significant amounts of substrate copurifywith the enzyme. HPLC-MS in positive ion mode (45) showedthat lanosterol copurifies with ScErg11p-6xHis in the absence ofadded ligand, supporting this interpretation (Fig. S1E).Electron density proximal to the heme iron, consistent with

the presence of an oxygen molecule coordinated to the hemeiron, fits with the ferrous dioxo intermediate predicted for theCYP51 reaction cycle. This feature also was seen in the crystal

Fig. 3. Sequence and predicted membrane interactions of ScErg11p. (A)Regions highlighted in yellow are predicted by OPM to lie inside the lipidbilayer. Red squares indicate acidic residues that were accessible to thewater-soluble glycinamide probe when ScErg11p-6xHis was incorporatedinto artificial liposomes. (B) Electrostatic surface map of ScErg11p with cut-away showing the primary and secondary vestibules. Surface electrostaticpotential is displayed with the molecular graphics software Chimera (59)with blue as positive and red as negative.

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structure without added ligand, which also had bound lanosterol(Fig. S1D). To interpret this peak, the electron density near theheme was assessed using crystallographic difference-map re-finement. Fo–Fc maps calculated after several rounds of re-finement showed that a single water (10 electrons) would generateinsufficient positive density, whereas imidazole (32 electrons)would give a strong negative density peak. Placing O2 (16 elec-trons) gave a flat Fo–Fc electron density map consistent with O2being at the heme site. Although unusual for P450s, this result isnot without precedent (46) and may be caused by reduction afterX-ray radiolysis (47) or purification of ScErg11p-6xHis trappedin the oxygen-bound state. The distance of ∼8 Å between oxygenand C14 methyl is consistent with a lanosterol binding in a pre-catalytic rather than a catalytically active state. Visual inspectionof the electron density maps and a high real-space R-factor in-dicated the need for considerable care in placement of the ligandin the lanosterol costructure. Minimally biased Fo–Fc electrondensity omit maps, calculated with lanosterol omitted to placethe lanosterol correctly, resulted in a ligand placement withreasonable correlation coefficient (0.813). Bound lanosterol isoriented differently from other lanosterol-like molecules in re-lated CYP51s (24), and the electron density offers no evidence ofthese alternate orientations.Density consistent with a second lanosterol-like molecule

projects from the secondary vestibule (Fig. 5 A and B). In allScErg11p structures examined, a hydrophobic tail fills the nar-row channel from the secondary vestibule to the surface ofErg11p, whereas a multiring head that projects into solutionappears to lack the C14 methyl group. Lack of density for theC14 methyl group suggested that the lanosterol-like moleculewas the reaction product 4,4-dimethyl-cholesta-8,14,24-trienol ora subsequent metabolite that likely copurified with the enzyme.Mass spectrometry of the purified ScErg11-6xHis detected lan-osterol, larger quantities of zymosterol, and traces of 4,4-dimethyl-cholesta-8,14,24-trienol (Fig. S1E). If a product egress chan-nel exists, the narrow channel off the secondary vestibule mayprovide a selectivity filter for the product; that is, removal of theC14 methyl group of lanosterol may be required for the multiringhead group to traverse the channel (Fig. 5B). Erg11p has beenshown to interact physically with Erg25p-Erg27p and possiblywith Erg24p (48). Presentation of 4,4-dimethyl-cholesta-8,14,24-

trienol on Erg11p then would allow concerted reduction byErg24p (C14 sterol reductase) and demethylation at carbon 4by Erg25p (C4 sterol methyl oxidase), Erg26p (C3-sterol de-hydrogenase), and Erg27p (3-keto sterol reductase) in complexwith the scaffold protein Erg28p to give zymosterol in the er-gosterol biosynthesis pathway (48). Alternatively, the retentionof 4,4-dimethyl-cholesta-8,14,24-trienol and zymosterol by Erg11pmay play roles that include conferral of structural stability ora feedback control mechanism. Two regions of weak excesselectron density located near the heme and adjacent to K220bordering helix G are likely hydrophobic molecules, possiblydetergent or phospholipid copurified with the enzyme, (Fig.S2 A and B).

DiscussionAzole Binding and Antifungal Resistance. Our structures identifyimportant ligand-binding constraints. The antifungal triazoledrug ITC extends from the active site to just beyond the mouthof the entry channel, similar to posaconazole in the T. bruceiCYP51 structure (49). The triazole group of the ITC makes acoordination bond with the heme iron. The space occupied byITC (Fig. 4B) fits closely with that occupied by lanosterol andbound O2 (Fig. 4A), with the triazole head group displacing theO2 and the di-halogenated headgroup replacing the first sterolring. The expected chlorine atoms on the dichlorophenyl groupconsistently showed strongly negative Fo–Fc density, suggestingthat the chloro groups had been damaged through photoreduc-tion upon X-ray data collection. As a result, the chloro groupshave been refined to zero occupancy in the model. In confir-mation of our findings, the triazole head groups in the smallertriazole drugs FLC and VCZ coordinate with the heme iron anddisplace the presumed O2, and their unmodified difluorinatedhead groups fill the space occupied by the first sterol ring oflanosterol and the sterol analog estriol (Fig. 4 A and B and Fig.S1 A–C). The long tail of ITC fills the entry channel and appearsto exclude all but one water molecule. The tail of ITC lies withina hydrophobic pocket lined by helices F and F′ (F236, P238, andF241), consistent with previous structures of truncated CYP51family members (50), and by the residues L380HSL383 andF506TSMV510 (Fig. 4B and see Fig. S4A).

Fig. 4. Lanosterol and ITC binding in ScErg11p. (A) Lanosterol is depicted with carbon atoms colored cyan and heme with carbon atoms colored yellow. Selectedoxygen atoms are colored red, nitrogen blue, sulfur gold, and iron brown. Electron density is depicted as a simulated annealing 2Fo–Fc omit map contoured to0.7σwith the ligand, heme, and oxygen omitted from the electron density calculation. A bond links the heme–oxo complex. (B) ITC in a coordination complex withactive-site heme. The color scheme is as in A, with ITC carbons colored magenta. Electron density is depicted as an Fo–Fc map contoured to 2.5σ with the liganddensity omitted from the calculation. (C) Conserved amino acids in fungi that are commonly mutated in antifungal resistance are depicted with carbon atomscolored orange. Amino acids conserved in S. cerevisiae and in themain fungal pathogens of humans but not in human CYP51, and not mutated, are depicted withcarbon atoms colored green. Amino acids that are similar only in the pathogenic fungi are shown in cyan. Other atoms are colored as in A.

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Although less than 24% of the amino acids in ScErg11p areconserved among fungi (50), there is an especially high con-centration of conserved residues near the heme (Fig. S4A) andactive-site cavity. These conserved residues include the abso-lutely conserved coordination of the C470 sulfur to the hemeiron and the ionized K151, R385, and H468 side chains. Thesequence G310VLMG314 in helix I is a consensus sequence(GXXXG) for sterol (cholesterol) binding, with the carbonyl ofG310 making the only polar interaction with lanosterol and aslightly kinked face toward ligand.The structure of the active site and substrate channel may

explain the susceptibility of some azole-resistant clinical isolates,including Candida albicans mutants F126L, Y132F/H, P230L,G307S, and F380S, Aspergillus fumigatus G54E/F and I301,Cryptococcus neoformans Y145F, and Ajellomyces capsulatusY136F (51, 52) to ITC or FLC (Fig. S3). For example, Y140 inS. cerevisiae (Fig. S3C) is equivalent to Y132 in C. albicans, Y145in C. neoformans, and Y136 in A. capsulatus. The absence of theheme-binding hydrogen bond in S. cerevisiae Y140F/H mutationscould modify the orientation of the heme and reduce suscepti-bility to both FLC and ITC. In contrast, G73E/F mutations inScErg11p, equivalent to G54E/F in A. fumigatus Cyp51A, are inthe mouth of the substrate channel and might be expected tomodify susceptibility to the long-tailed triazoles ITC and pos-aconazole but not short-tailed VCZ.Numerous azole-resistance mutations occur outside the ac-

tive site. Significantly, the N22D azole-resistance mutation inTMH1 of A. fumigatus Cyp51A (ScErg11p N42D) supports theproposition that interactions between TMH1 and the catalyticdomain can affect active-site function. CYP51s from S. cerevisiaeand the fungal pathogens of humans (Fig. S4) contain a group ofconserved/similar aromatic residues (F60, Y61, W62, Y72, Y77,F79, F80, Y87, F90, H381) bordered by TMH1 that form a com-pact, buried structure. In concert with neighboring conserved/similar hydrophobic residues (V59, L95, L96 L383, L407) and thepolypeptide backbone (A69, G73, S382), this grouping supportsa narrow entry channel into the active site and egress vestibules.The tight vestibules orient the lanosterol and may provide gatedentry of demethylated reaction product into the egress channel.Finally, mutation conferring azole resistance at residues homol-ogous to ScErg11p L95, F241, H381, and S382 has yet to be re-ported. These residues along with the chemically similar residueI139 could provide fungal-specific side-chain contacts for the design

of novel antifungals directed against the major fungal pathogensof humans (Fig. 4C and Fig. S3B).

Bitopic Membrane Proteins. Full-length structures of bitopicmembrane proteins with one transmembrane helix are absent inthe Protein Data Bank, probably because of the difficulties withordered crystallization. This deficiency has limited understandingof how single-transmembrane helices orient enzymes/sensors atthe bilayer surface (1). The X-ray structure of ScErg11p-6xHis,a full-length fungal cytochrome P450 enzyme, shows that thetransmembrane domain not only tethers the protein to the ERbut also may orient it relative to the bilayer. The surprisingly rigidinteraction between the transmembrane domain and catalyticdomain may establish the pose required for catalysis and in-teraction with other protein partners such as Erg24p and Erg25p–Erg27p. With monospanning membrane proteins constitutingabout half of all membrane proteins, structural information sup-porting properties beyond tethering and localization in ScErg11pprovides clues about constraining polar residues in other mono-spanning enzymes. It also may define a motif that may be a featureof other membrane-bound enzymes.The ScErg11p structures identify lanosterol binding in the

active site of a CYP51, a substrate channel linking to the lipidbilayer, and a secondary vestibule with a product exit channel.The model also more fully maps how triazole antifungals interactwith a fungal cytochrome P450 to block catalysis and identifiespossible interactions that confer azole resistance applicable topathogenic fungal species. Like other CYP51 structures, the in-tact enzyme displays less conformational heterogeneity betweenstructures than seen in many truncated ectodomain eukaryoticcytochrome P450s, suggesting that the transmembrane domainitself or associated hydrophobic molecules such as lipids or re-action product may decrease conformational heterogeneity andprotect the active site from bulk water. The structures shownhere may enable the development of new molecular models tofacilitate drug design that targets fungal CYP51s or othereukaryotic cytochrome P450s and may provide a practical basisfor the design of therapeutics with minimized off-target effects.

MethodsS. cerevisiae ScErg11p was overexpressed homologously as a 6×His derivative(ScErg11p-6xHis) from the PDR5 locus in the host yeast strain ADΔ (Table S1)(55). ScErg11p-GFP expressed in ADΔ was found in nuclear-associated ER(Fig. S5A). ScErg11p-6xHis was purified from strain MMLY941 by extractionof a crude membrane fraction with the detergent DM, Ni-NTA-agarose af-finity chromatography, and size-exclusion chromatography (SEC) in thepresence or absence of ITC (Fig. S5B). The purified protein behaved asa detergent micelle-protein monomer during SEC, as is consistent witha molecular mass for the protein of ∼62 kDa (Fig. S2C). It bound ergosteroland showed type II binding of known antifungals (Fig. S5D). The purifiedprotein was authenticated by mass spectrometry of tryptic fingerprints atthe Stanford University Mass Spectrometry facility (Fig. S5E).

Red crystals were obtained initially by hanging-drop vapor diffusion at 18 °Cin 200-nL drops with a MemGold (Molecular Dimensions Limited) screen setusing a Mosquito robot (TTP Labtech). Crystallization conditions were opti-mized, and large, red, boat-shaped crystals (100 μm) were obtained in 2- to 6-μLdrops with the purified protein at ∼20 mg/mL in 10% (wt/vol) glycerol, 150 mMNaCl, 4× critical micellar concentration DM, and 20 mM Hepes (pH 7.5) mixed1:1 with 43–45% (wt/vol) PEG 400 in 100 mM glycine (pH 9.3–9.5) (Fig. S5F).

Complete datasets were obtained for crystals at the Berkeley AdvancedLight Source BL 8.3.1 with a beam at wavelength 1.1587 Å (Table S2). Datawere processed, integrated, and scaled using the HKL2000 (53) package. Aninitial model was determined by molecular replacement using the solubledomain of the HsCYP51 model 3LD6 (20) in Phaser (54). Refinement wasperformed using both Refmac5 in the CCP4 (55) suite of programs andPhenix (56), and model building was performed using Coot (57). Modelvalidation was performed in Phenix and with Molprobity (55) and PROCHECK(58). Display of electrostatic maps was performed in Chimera (59).

ACKNOWLEDGMENTS. We thank James Holton and George Meigs at beam-line 8.3.1 of the Advanced Light Source at Lawrence Berkeley National

Fig. 5. Multiple ligands bind to ScErg11p. (A) Electron density of lanosterol-like molecule. Fo–Fc density contoured to 2.0σ is shown in green witha theoretical model of 4,4-dimethyl-cholesta-8,14,24-trienol placed. The 4,4-dimethyl-cholesta-8,14,24-trienol is depicted with carbon atoms coloredcyan. (B) Hypothetical model of ligand binding with surface representationof the primary and secondary vestibules. Lanosterol bound in the active siteis depicted with carbon atoms colored cyan. The second lanosterol-like mol-ecule is depicted with carbon atoms colored purple. Oxygen atoms are col-ored red, nitrogen blue, and iron brown. The outside surface of the vestibulesand active site are depicted as a light mesh surface colored dark gray, andtheir visible inside surface is colored light gray.

Monk et al. PNAS | March 11, 2014 | vol. 111 | no. 10 | 3869

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Laboratory; Bill Harries, Rebecca Robbins, and Erwin Lamping for sup-port; Paul Ortiz de Montellano and John Cutfield for critical reading; andAvner Schlesinger for helpful computational suggestions. This research was

supported by National Institutes of Health Grants U54GM094625, GM24485,and GM073210, the Marsden Fund of the Royal Society of New Zealand, andthe Otago Medical Research Foundation.

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3870 | www.pnas.org/cgi/doi/10.1073/pnas.1324245111 Monk et al.

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