Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane:...

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Archives of Biochemistry and Biophysics 468 (2007) 82–91

ABB

Mapping of cytochrome P450 2B4 substrate binding sites byphotolabile probe 3-azidiamantane: Identification of putative

substrate access regions

Petr Hodek a,*, Martin Karabec a, Miroslav Sulc a,c, Bruno Sopko d, Stanislav Smrcek b,Vaclav Martınek a, Jirı Hudecek a, Marie Stiborova a

a Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 40 Prague, Czech Republicb Department of Organic and Nuclear Chemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 40 Prague, Czech Republic

c Institute of Microbiology v.v.i., Academy of Science, Videnska 1083, 142 20 Prague, Czech Republicd Department of Natural Sciences, Faculty of Biomedical Engineering, Czech Technical University in Prague, Sitna Sq. 3105, 272 01 Kladno, Czech Republic

Received 2 July 2007, and in revised form 12 September 2007Available online 29 September 2007

Abstract

To investigate structure–function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labelingof rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide

I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected intoCYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel bindingregions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the activesite from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data ofphotoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.� 2007 Elsevier Inc. All rights reserved.

Keywords: CYP2B4; Photoaffinity labeling; Diamantane; Access channel; Enzyme structure

Cytochromes P450 (CYP)1 belong to the most exten-sively studied enzymes involved in metabolism of foreigncompounds (xenobiotics) such as drugs, pollutants, dyes,carcinogens, as well as endogenous compounds (steroidhormones, fatty acids, prostaglandins) [1]. Since a largenumber of hemoproteins of this gene superfamily areinvolved in carcinogen activation, the research of struc-ture–function relationships of CYPs is aimed at an under-

0003-9861/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2007.09.017

* Corresponding author. Fax: +420 221951283.E-mail address: [email protected] (P. Hodek).

1 Abbreviations used: CID, collision-induced dissociation; CYP, cyto-chrome P450; DIA, diamantane; DIA-N2, 3-azidiamantane; DMSO,dimethyl sulfoxide; HAP, hydroxylapatite; 3H-DIA-N2, tritium labeled 3-azidiamantane; MALDI-TOF, matrix assisted laser desorption/ioniza-tion-time of flight; PDB, protein data bank; TFA, trifluoroacetic acid;TPCK, 1-chloro-3-tosylamido-4-phenyl-2-butanone; 3D, three-dimensional.

standing of the molecular basis of the first stages ofcarcinogenesis. To shed light on this issue, it is essentialto know the three-dimensional (3D) structure of CYPs indetail. Besides bacterial cytosolic CYPs, several 3D struc-tures based on X-ray crystallography are available formembrane CYPs of mammalian origin [2].

Rabbit CYP2B4 serves as a prototype microsomal CYPof family 2, metabolizing mainly bulky hydrophobic xeno-biotics. This isoform has been used as an object of numerousmetabolic and structural studies. Recently crystals of N-ter-minal modified CYP2B4 chimeras, both of the substrate-free form and complexes with ligand inhibitors were pre-pared, and 3D structure models developed [3–5]. However,the problem remains to be solved how the substrate reachesthe active site deeply buried in the CYP macromolecule.Likewise, the membrane topology of CYPs, not entirelyunderstood as yet, should be investigated. One may expect

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P. Hodek et al. / Archives of Biochemistry and Biophysics 468 (2007) 82–91 83

that hydrophobic substrates, after concentration in themembrane bilayer, enter the access channel towards theenzyme active site. In addition, the reaction product (usu-ally less hydrophobic) should leave the active site to allowbinding of the next substrate molecule. It is unclear whetherCYP uses the same route for access of substrate and exit ofproduct. Potential routes of access and egress for substratesand products of CYP were established from moleculardynamics simulations of crystal structures of CYPs. Threechannels (1, 2, and 3; see Fig. 1) were identified as spatiallyand structurally distinct routes [6]. Trajectories in the regionof channel 2 were clustered into subclasses 2a–2f accordingto the secondary structure elements lining the ligand path-way as it emerges from the protein surface [7]. Pathway2a, in which the ligand passes between the F–G loop, B 0

helix/BB 0 loop/B–C loop, and the b1 sheet, was found inbacterial CYPs [8]. The most common pathway for themammalian CYPs (e.g., for CYP2C5) seems to be the egresspathway 2c, in which the ligand passes between the G- andI-helices and the B 0 helix/B–C loop [9].

In addition to molecular simulation, modeling and othercomputational approaches, chemical modification, spec-troscopy, and site-directed mutagenesis techniques havebeen employed to localize and examine the substrate accesschannels [10–16]. Among chemical modification techniquesthe photoaffinity labeling is useful for study of proteinstructure (for review, see [17]). It proved to be helpful formembrane-bound CYPs, difficult to examine by other tech-niques. Azides, diazirines, and benzophenones are mostfrequently introduced into the substrate molecule, provid-ing by photolysis the highly reactive intermediates forlabeling of substrate binding site. Several photolabile sub-strates have been used in the past as effective photoaffinityligands of mammalian and bacterial CYPs [18]. Recently,CYP putative access channels [14,19] as well as the aminoacid residues in their active sites [20,21] were identified byphotoaffintity probes.

In the present study, we examined substrate bindingsites of CYP2B4 by photoaffinity labeling with 3-azidi-

Fig. 1. Cartoon of CYP active site showing location of three classes ofsubstrate (ligand) access/egress pathways relative to the heme, I-helix andthe membrane. The heme plane is perpendicular to the viewer. Adoptedfrom Ludemann et al. [6] and Cojocaru, et al. [7].

amantane (DIA-N2), a photolabile derivative of theCYP2B4 substrate diamantane. The labeled tryptic pep-tides were separated on a C18-HPLC column and identifiedby amino acid sequencing or mass spectrometry. Theexperimentally defined binding sites of diamantane werecompared with results of computational docking experi-ments and with those of the known crystal structure ofgenetically engineered CYP2B4.

Materials and methods

Materials

Photolabile probe, 3-azidiamantane, was synthesized according toVodicka et al. [22]. A tritium labeled 3-azidiamantane (3H-DIA-N2) wasprepared as described by Hodek and Smrcek [23]. The specific radioac-tivity was 154 mCi/mmol. All chemicals used were of analytical grade orbetter.

Analytical methods

Protein concentration was determined by the method of bicinchonine-Cu+ chelation using bovine serum albumin as the standard [24]. Cyto-chrome P450 content was measured in 1-cm optical path cuvettes on aSpecord M-40 spectrophotometer (Carl-Zeiss, Jena, Germany) using areduced cytochrome P450 complex with CO and calculated according toOmura and Sato [25].

Cytochrome P450 2B4 preparation

Cytochrome P450 2B4 was purified from the liver of a rabbit treatedwith phenobarbital basically as described by Haugen and Coon [26]. Thefinal electrophoretically homogenous preparation had a specific content12.2 nmol of CYP/mg protein.

Photoaffinity labeling

CYP2B4 (60 nmols) diluted with 0.1 M potassium phosphate buffer(pH 7.4), containing 20% glycerol, to a final concentration of 1 nmol/mlwas incubated 20 min with equimolar 3-azidiamantane, DIA-N2 or 3H-DIA-N2. For the competition experiments, an equimolar amount of theCYP2B4 substrate, diamantane (DIA), (1 mM stock solution in DMSO)was added. The probe was photoactivated by a 1000 W Xenon lampequipped with a water hot trap at a distance of 25 cm for 2 min or in awater cooled photolyzer equipped with a mercury arc lamp RVK 125 W(Tesla Praha, Czech Republic) at a distance of 2.5 cm for 15 min. Reactionmixtures of CYP2B4 were irradiated in a Pyrex tube to shield from shortwave UV irradiation. Prior to trypsin digestion, non-covalently boundtritiated photoproducts were removed from CYP2B4 by chromatographyon hydroxylapatite (HAP) as follows. Dialyzed CYP2B4 samples wereapplied to columns (12 · 44 mm) equilibrated with buffer: 5 mM sodiumphosphate (pH 7.4) containing 20% glycerol and 0.2% Emulgen 911. Thecolumn was extensively washed with the equilibration buffer until radio-activity in the eluate reached background values. Then, the detergent wasremoved from the column by extensive washing with 5 mM sodiumphosphate buffer (pH 7.25) containing 20% glycerol. The CYP2B4 cova-lently labeled with radioactive probe was eluted from the column of HAPwith 0.4 M potassium phosphate buffer (pH 7.25) containing 20% glycerol.

Trypsin digestion

The labeled CYP2B4 samples were dialyzed against 0.2 M ammoniabicarbonate buffer (pH 8.3), containing 0.1 mM CaCl2 and 2% glycerol.Digestion was performed by 1-chloro-3-tosylamido-4-phenyl-2-butanone

84 P. Hodek et al. / Archives of Biochemistry and Biophysics 468 (2007) 82–91

(TPCK) treated trypsin (CYP/TPCK trypsin ratio 1:44). After incubation(6 h at 37 �C), a second treatment with the same amount of TPCK trypsinwas added and the incubation continued for an additional 16 h. Thedigestion was stopped by addition of TFA to 1% of final concentrationand the sample was concentrated in a Speed Vac Concentrator SVC 200H(Savant, USA) for 5 h. The resulting residue in the tubes was stored at�20 �C for HPLC analysis.

HPLC separation of peptides

All experiments with radiolabeled CYP2B4 were carried out with aPerkin-Elmer Series 4 HPLC system equipped with a UV–VIS detectorLC-95. Samples were applied to a 200 ll loop of Rheodyne columninjector connected to a RP-C18 lBondapak 4.6 · 250 mm column (WatersCorporation, Milford, MA). The tryptic digest was extracted in 10%acetonitrile with 1% TFA. Tryptic peptides were separated on a C-18column using a linear gradient of acetonitrile (0–75%) within 62.5 min inthe presence of 0.1% TFA with a flow rate of 1 ml/min. Based on radio-activity and 214 nm absorption, fractions of the elution profile werepooled and these pools re-chromatographed on a C-18 column undersimilar conditions (a linear gradient of acetonitrile 0–75% within 62.5 min,flow rate 1 ml/min) except for the presence of 0.1% ammonium acetate(pH 6.0). To obtain the homogeneous radioactive peptide fraction forsequencing, a further chromatography using a shallower acetonitrile gra-dient (0.5%/min) was undertaken. Pure radioactive peaks were subjectedto Edman N-terminal sequencing on a Protein Sequencer LF3600D(Beckman Instruments, USA) according to the manufacturer’s manual.

Similarly, CYP2B4 labeled with non-radioactive DIA-N2 was cleavedby TPCK-trypsine and peptides were separated on a Dionex HPLC systemwith a RP-C18 semi-preparative column (SynChropak RPP-100,250 · 10 mm, MICRA Scientific, Inc., USA) under the same conditions asmentioned above. A HPLC system equipped with UV–VIS detector (UVD170S/370S) was operated using Chromeleon software (ver. 6.11). Thepeptide elution pattern was monitored at 214 and 280 nm. Fractions (30 s)of selected regions of the elution profile were dried and analyzed byMALDI-TOF (see below).

MALDI-TOF analysis of the labeled CYP2B4

MALDI-TOF analyses were performed on a Bruker-Biflex (Bruker-Daltonics, USA) equipped with a 337 nm nitrogen laser and a delayedextraction ion source. As a MALDI matrix, a 10 mg/ml solution of a-cyano-4-hydroxycinnamic acid in aqueous 30% acetonitrile and 0.1% FA(v/v) was used. Positive-ion mass spectra of peptide maps were collected inthe reflection mode. The sample (1 ll) was loaded on the target, allowed todry at ambient temperature and covered with the matrix solution (1 ll).Spectra were externally calibrated by employing the monoisotopic(M+H)+ ion of a peptide standard (human angiotensin I). Every recov-ered spectrum was compared with theoretical peptide maps and fractionswith ghost peaks were additionally analyzed by LC–MS/MS.

LC–MS/MS analysis of the labeled CYP2B4

The diluted and MALDI-TOF analyzed HPLC fractions were loadedon a LC–MS/MS system (LCQDECA, ThermoQuest, San Jose, CA) andtandem mass spectra were acquired. Five microliters of the HPLC fraction(previously analyzed on the MALDI-TOF) were applied on the capillarycolumn (0.1 · 150 mm) packed with 10 cm of C18 RP resin MAGIC AQ(MichromBioresources, USA). Peptides were separated using gradientelution: 65 min from 5% acetonitrile/0.5% acetic acid to 40% acetonitrile/0.4% acetic acid, 15 min from 40% acetonitrile/0.5% acetic acid to 70%acetonitrile/0.4% acetic acid at flow rate of 1 ll/min. The column wasdirectly connected to an LCQDECA ion trap mass spectrometer (Ther-moQuest, San Jose, CA) equipped with a nanoelectrospray ion source.Spray voltage was held at 1.2 kV, tube lens voltage was 30 V. The heatedcapillary was kept at 175 �C with a voltage 10 V. Collision energy was keptat 42 U and the activation time was 30 ms. Positive-ion full scans were

acquired over an m/z range 350–1600. Collisions were done for the topthree most intense ions in each full MS scan. Dynamic exclusion wasenabled with repeat count of 2. Selected collision spectra were manuallysearched with respect to CYP2B4 sequences and potential modifications.

Docking of diamantane into CYP2B4 structure

Diamantane was docked into the CYP2B4 crystal structures (1PO5,1SUO, 2BDM in PDB code) [3–5] and the homology model describedpreviously [14]. Briefly, for the docking Autodock 3.05 software wasemployed, using the ‘‘genetic’’ algorithm method (27,000,000 generations,200 populations) using 30 runs for the diamantane substrate. First, thewhole CYP2B4 molecule was used as a target (the mesh point distance was5 nm). Then, potential sites were ‘‘refined’’ with the mesh point distance of1.5 nm. The conformation having the lowest energy was selected as theresult. Values of apparent dissociation constants (Kd) were determinedusing program Autodock 3.0.5 [27].

Results

Incorporation of [3H]-3-azidiamantane into CYP2B4

In order to identify the CYP2B4 regions involved indiamantane interaction tritium labeled 3-azidiamantane(3H-DIA-N2) was used. In pilot experiments, the bindingof the diamantane probe to CYP2B4 and the yield of label-ing were determined. CYP2B4 was photolyzed in the pres-ence of 3H-DIA-N2 or pre-photolyzed 3H-DIA-N2 andthen the non-covalently bound probe was removed fromthe precipitated protein by washing with acetone. The spe-cific content of radiolabel was 316 DPM/lg of CYP2B4protein, compared with 1.1 DPM/lg for CYP2B4 irradi-ated with the pre-photolyzed probe. Based on the proberadioactivity added into the reaction mixture, the yield oflabeling was determined to be 10.8%. Labeling of CYP2B4with 3H-DIA-N2 on a preparative scale (60 nmol CYP2B4,60 nmol 3H-DIA-N2) was carried out and non-covalentlybound probe was removed on a HAP column. The result-ing sample contained 2.2 · 104 DPM/nmol CYP2B4, repre-senting a 9% yield of probe incorporation. The yields ofCYP2B4 covalent labeling, determined in pilot and pre-parative experiments, proved the efficacy of the probe car-bene to incorporate into the target protein.

CYP2B4 labeled with 3H-DIA-N2 and purified by chro-matography on HAP was treated with trypsin and theobtained peptides were separated by HPLC. All fractionswere analyzed for their radioactivity and the fractions oftwo radioactive regions (A, B eluted within 30–45 min,Fig. 2) were pooled. The mobility of tryptic peptideslabeled with DIA was shifted towards longer retentiontimes, obviously reflecting the hydrophobicity of theattached probe. Radioactive pools A and B containing18.2% and 27.4% of applied radioactivity, respectively,were re-chromatographed and the purified radiolabeledpeptides sequenced. The sequencing data and positions oflabeling in the CYP2B4 protein are summarized inTable 1. In pool A, two re-chromatographed fractionsrevealed radioactive fragments. The first fraction, referredto as peptide I (A1 in Table 1), contained a labeled frag-

Fig. 2. Elution profile of tryptic peptides of CYP2B4 labeled with 3H-DIA-N2. HPLC separation was carried out on an analytical C18-column using 0–75% acetonitrile gradient in 62.5 min. Based on the radioactivity profile, the fractions of indicated time intervals A and B were pooled for further peptidepurification.

Table 1CYP2B4 tryptic peptides labeled with the photoaffinity probe

Labeled fragment Elution time (min) Re-chromatographya Fragment sequence CYP2B4 position

Peptide I 30.0–38.0 (A)b A1 LGDLIPFGVPHTVTK 359–373Peptide II 38.0–46.0 (B) B1 LPPGPSPLP 30–38

B2 LPPGPSPLPVLGNLLQM*R 30–48B3 LPPGPSPLPVLGNLL*MDR 30–48B4 LPPGPSPLPVLGNLLQM*R 30–48B5 LPPGPSPLPVLGNL*QM 30–46B6 *PPGPSPLPVLGNL*Q 30–45B7 LPPGPSPLPVLGNLLQM*R 30–48

Peptide III 34.0–34.5 (A) NA FSLATMRDFGMGKR 127–140Peptide IV 34.0–34.5 (A) NA RICLGEGIAR 434–443

Peptides I, II were analyzed by Edman N-terminal sequencing and peptides III, IV by MALDI-TOF. Re-chromatographed radiolabeled fractions of poolB (eluted between 38–46 min) contained tryptic peptide II, and the asterisks mark position of unidentified amino acids, presumably modified withdiamantane. The underlined amino acid residue (R133) of peptide III was found to be modified with the probe.

a Fraction labels (numbers) refer to the re-chromatography peak order. NA, not applicable.b Letters in parentheses denote the radioactive pools used for re-chromatography.


ment matching the sequence position Leu359-Lys373.Sequencing of the second fraction (A2, not shown) resultedin sequences of three peptides (Leu70-Arg85, Tyr401-Lys421,Ile435-Arg443) but the label location was not identified.However, it is noteworthy that the peptide Ile435-Arg443

was identified also by MALDI as labeled peptide IV (seeTable 1 and results shown below).

The pool B, peptide II, comprised seven fractions (B1-7)containing tryptic fragments, starting solely at Leu30, withthe probe incorporated in various positions of the peptide(see Table 1).

Photoaffinity labeling of CYP2B4 by 3-azidiamantane

To demonstrate the specificity of the labeling ofCYP2B4 at substrate binding site(s), additional experi-

ments were carried out using non-radioactive 3-azidiaman-tane (DIA-N2) in the presence or absence of competingsubstrate, diamantane (DIA). Under these conditions, theextent of specific labeling should be significantly reducedby competitive binding of DIA and DIA-N2. Trypticdigests of CYP2B4 photolyzed alone (sample 1), andCYP2B4 photolyzed with DIA-N2 in the absence (sample2) or presence of DIA (sample 3) were prepared and sepa-rated under identical conditions by preparative C18-HPLC(see Fig. 3 for sample 1). As found in the experiments uti-lizing 3H-DIA-N2 described above, the highest levels ofradioactivity were eluted within 30–46 min (Fig. 2). Hencethe fractions (30 s) of this elution time span were collected(Fig. 3). Fractions of all three CYP2B4 samples (1–3) weresubjected to MALDI-TOF analysis. Comparing theMALDI-TOF mass spectrometric data of corresponding

Fig. 3. A representative chromatogram of the trypsin digest of the photolyzed CYP2B4. Peptides were separated by preparative C18-HPLC using 0–75%acetonitrile gradient. Fractions (30 s) of the marked region were collected. Corresponding fractions of three individual samples (photolyzed CYP2B4,photolyzed CYP2B4 with DIA-N2, photolyzed CYP2B4 with DIA-N2 and DIA) were compared using MALDI-TOF.


fractions, masses of specific adducts (tryptic pep-tide + DIA) were searched for. Only fractions collectedwithin 34.0–34.5 min of CYP2B4 sample 2 contained peaks(m/z 1274 and 1804) that were absent in the CYP2B4 sam-ples 1 and 3 photolyzed without DIA-N2 and with protec-tive DIA, respectively. Masses of these fragments (seepattern in Fig. 4) did not agree with any of the theoreticalCYP2B4 digest fragments, however, they match exactly themasses of tryptic peptides of CYP2B4 modified with theprobe carbene (186.3 Da). The first tryptic fragment, pep-tide III (see Table 1), corresponds to the CYP2B4 sequencePhe127-Arg140 (m/z 1803.4 = 1616.8 + 186.3). An oxidizedform of this fragment (m/z 1819.3 = 1616.8 + 16 + 186.3)was identified, as well (Fig. 4). The spectrum of thesecond peak was consistent with the tryptic fragment,peptide IV, spanning the sequence Arg434-Arg443

(m/z 1273.3 = 1086.6 + 186.3).The covalent structure of suspected adducted peptides of

the peptide III was determined using tandem MS (MS/MS).Daughter ion spectra of peptide III fragmented by colli-sion-induced dissociation (CID) are shown in Fig. 5. Dataconfirmed the deduced amino acid sequence of the labeledpeptide and indicated Arg133 to be covalently modified withthe diamantane moiety. In addition, a CID spectrum ofoxidized peptide III showed the characteristic loss of meth-anesulfenic acid from the side chain of oxidized Met132

(Figs. 4 and 5).

Docking experiments

To localize putative diamantane binding site(s) withinthe CYP2B4 structure, docking experiments were carriedout. The crystal structures (1PO5, 1SUO, 2BDM in PDBcode) and the model of CYP2B4, developed by us previ-ously [14], were used. The lowest value (1.7 · 10�6 M) of

an apparent dissociation constant (Kd) for CYP2B4-DIAbinary complexes was determined for the DIA binding intothe active site in the model structure (see Fig. 6a). AnotherDIA-high affinity binding site (Kd = 3.4 · 10�7 M for themodel structure) was found outside the active site, on theCYP2B4 surface. In this second binding site, the DIA mol-ecule is accommodated in the cavity lined with B–B 0/B 0–Cloops and b strand 1–4 (see Fig. 6b).

Discussion

Although CYP enzymes have been studied for almostfive decades, and several crystal structures of CYPs havebeen obtained, the structure–function relationships ofCYPs are poorly understood based on crystal structuresalone. CYP chimeras are frequently used for X-ray crystal-lography to facilitate their expression and crystallization.The membrane-anchor peptide has to be removed in mostcases and other sequence alterations (e.g., FG loop/G helixof CYP2C5) are often needed to prevent dimer formation(also for CYP2B4 crystal [3]). The major advantage ofphotolabeling in comparison to X-ray crystallography con-sists of gaining insights into the protein structure of ‘‘wild’’(not mutated or shortened) enzymes in their native envi-ronment (membrane/water). Thus, together with otherapproaches, e.g., crystallography and molecular dynamicssimulations [6], chemical modification techniques (e.g.,applying photoreactive substrates) provide the potentialfor refining the architecture of native CYPs.

Using the skeleton of a highly selective substrate ofCYP2B4, diamantane, a photoaffinity probe, 3-azidiaman-tane, was prepared. First, we should consider the generalproperties of the photoaffinity label and its mode of bind-ing to the CYP2B4. Our previous experiments [28] haveshown that after photoactivation, the compound reacts

Fig. 4. MALDI-TOF analysis of tryptic peptides of photolyzed CYP2B4. Panels show masses of HPLC fractions (34.0–34.5 min) for photolyzed CYP2B4(a), and for photolyzed CYP2B4 with DIA-N2 (b). The highlighted masses m/z 1273.3, m/z 1803.4, and m/z 1819.3 indicate peptides specifically labeledwith diamantane. In the presence of DIA during the photolysis the formation of these adducts was prevented by the competing non-reactive DIA.

Fig. 5. Schemes of peptide III fragmentation based on the CID daughter spectrum of m/z 1819.3 species. The calculated theoretical mass of b7 ion and allfollowing ions from b-series have m/z values with difference of 186 Da, indicating the covalent attachment of the probe to Arg133. The fragmentationscheme also shows the detected ions with the loss of methanesulfenic acid (�64 Da) from oxidized Met132 (given in italics). The m/z values of b10 ion oflow peak intensity are shown in parentheses.


Fig. 6. CYP2B4 model structures with the docked DIA molecule (ingreen) in the active site (a), and in the outer region (b). Labeled peptide I

(Leu359-Lys373) is shown in black. Images were generated using PyMOL[35].


readily with hydrocarbons (hexane). At the same time, theelectrophilic carbene intermediate formed by photolysisreacts preferentially with the electron-rich compounds(alcohols, piperazine, water, dioxygen), even when presentas traces. Taken together with the short lifetime of the car-bene intermediate, this effectively precludes a possibilitythat the activated label would ‘‘migrate’’ from the bulk sol-vent towards the CYP molecule (or along the protein sur-face). Therefore, the sites labeled in the enzyme areidentical with the location, where the label was boundbefore the photoactivation.

Our previous studies dealing with interaction of thediamantoid label with CYP molecules have shown thatthe compound binds with a high specificity to the sub-strate binding site, and the introduction of photolabilediazirine group does not change the binding propertiessignificantly [23,28]. This was proven both by the valuesof spectroscopic dissociation constant (indicating theextent of low to high spin conversion upon binding)

and the inhibitory effect of the label on CYP enzymaticactivity.

In the present study, photoaffinity labeling of CYP2B4with photoaffinity probe, 3-azidiamantane, revealed fourtryptic peptides of CYP2B4 modified with the carbene ofDIA (see Table 1). We compared our experimental datawith the crystal structures for the substrate-free CYP2B4(PDB code 1PO5), and for the CYP2B4 with bound inhib-itors, 4-(4-chlorophenyl)imidazole (CPI) (PDB code1SUO), and bifonazole (BF) (PDB code 2BDM). Theinhibitor-bound structures differ significantly both fromeach other as well as from the substrate-free CYP molecule.In addition, whereas both CPI and BF react as ligands tothe heme iron in CYP2B4, diamantane and 3-azidiaman-tane are bound as substrates [28]. Therefore, we employedthe CYP2B4 model structure [14] additionally to the crystalstructures, and the same model was used also to show puta-tive DIA binding sites within the enzyme 3D structure.

Peptide I

The tryptic peptide I (Leu359-Lys373) is located betweenthe K–K 0 helices. It goes through the active site parallelto the heme plane and protrudes into the protein surface(see Fig. 6). Since the peptide sequencing of the labeled pep-

tide I did not identify the DIA modification site(s), dockingexperiments with CYP2B4 model [14] were carried out toaddress the question of the binding areas of DIA in thisregion. Interestingly, as shown in Fig. 6, the docking ofdiamantane resulted in two DIA binding sites connectedby the peptide I. The first site is located in the enzyme activesite (see Fig. 6a). There, the docked DIA makes the closestcontact with Val367 (3.5 A) of peptide I. The second pre-dicted DIA-binding site is on the CYP2B4 surface (seeFig. 6b) where the closest residue of peptide I to the dockedDIA molecule is Thr372 (3.0 A

´). Although a second DIA-

binding site has not been previously identified for CYP2B4,a similar outer substrate binding site was identified inCYP101 with another photoaffinity probe [29]. The labeledMet103 of CYP101 is localized in the B 0–C loop, the seg-ment corresponding to the outer region where DIA dockedin CYP2B4. The assumed labeling of CYP2B4 on its sur-face can be inferred from experiments with 2-adamantanediazirine [30]. Upon binding of this adamantane photo-affinity probe, the authors indicate the presence of watermolecule(s) in the active site which prevents the covalentbinding of activated probe to amino acid residues. Undersuch conditions the probe carbene is quenched by captureof a water molecule, resulting in 2-adamantanol formation.

In addition, we compared our results with the data fromrecent molecular dynamics simulations. The position ofdocked DIA (peptide I, see Fig. 6b) marks the entranceto the substrate channel 2b/2e proposed for CYP2B4 [7]and channel 2 in the CYP2B1 active site [31]. Based onrecent knowledge obtained of the membrane topology ofmammalian CYPs, the labeled region of CYP2B4 aroundthe docked DIA (peptide I, see Fig. 7) is either embedded

Fig. 7. CYP2B4 embedding in the membrane. Expected position of the membrane indicated by the filled rectangle. Shade intensity estimates the depth ofprotein penetration. Front (a), and rear (b) views show positions of labeled peptides I, III, IV. The docked DIA is shown near the B 0 helix. Adopted fromZhao et al. [5].


[5] or in close proximity to the membrane [7,10,12,32]. It ispossible that CYP2B4 adopts an orientation in the mem-brane that allows immersion of the channel opening intothe membrane bilayer, as well as exposure to cytosol.

Fig. 8. Insight into the ‘‘door structure’’ of the proposed substrate accesschannel with DIA in the active site. The opening to the channel is formedfrom the DIA labeled pre-helix A peptide II (arrows point to the DIAmodification sites), hairpin structure (tip residue Val 476 is shown), and F/G loop of the CYP2B4 structure (PDB code—2BDM).

Peptide II

The tryptic peptide II of CYP2B4, occurring in fractionsof the pool B, covers the region Leu30-Arg48 that contains amajor part of the proline rich region preceding the A-helix.Wide distribution of this sequence among fractions of poolB was caused by incorporation of diamantane at severalsites. The extensive labeling of several amino acid residuesof CYP2B4, mainly at the C-end of the pre A-helix region,suggests this region to be highly exposed to photoaffinityprobe. Hence, this identified peptide is apparently a partof an important region for substrate entrance to the buriedactive site. Alternatively, we cannot exclude that the exten-sive labeling of several amino acids may reflect a regionembedded in the membrane, which posses a high effectiveconcentration of the label.

Nevertheless, our data are in agreement with the pre-sumed binding of CYP2B4 in the endoplasmic reticularmembrane. According to recent views of the membranetopology of microsomal CYPs, these enzymes are attachedto the membrane by one hydrophobic N-terminal helix,hydrophobic regions dipping into the lipid bilayer and bycharged residues that interact with the phospholipid sur-face [5,10,12,32]. Hydrophobic surfaces at the CYP bottompenetrating into the membrane are formed by residues 30–45 before helix A, the b1-1-b1-2 loop, residues 101–116before helix C, helix F 0, and flanking residues. The locationof the labeled peptide II (mainly its second half) suggeststhat it is part of the substrate access channel.

The pre A-helix region, together with the F–G loop,was suggested by Dai et al. [10] to form the ‘‘door struc-ture’’ of the access channel. Indeed, the F–G region ofCYP2B4 was previously identified by us, using another

photoprobe (aminoazido-desmethylbenzphetamine), asthe opening of the access channel [33]. As shown inFig. 8, the structures identified with photoaffinity probes,together with residues 476–478 (b hairpin) determine theentry into the substrate access channel. This substrateentry route was proposed also by Zhao et al. [5] andshares similar location with substrate egress trajectory 2fas characterized by molecular dynamics simulations inmammalian CYPs [7].

Fig. 9. View of the heme proximal side of CYP2B4 model structure.Labeled peptides III (Phe127-Arg140) and IV (Arg434-Arg443) belonging tohelices C and L, respectively, are shown in black. The putative DIAbinding site is located between C-terminal part of C-helix and N-terminalpart of L-helix. The C-terminal part of C-helix contain Arg133 adductedwith DIA carbene.


Peptides III and IV

Another DIA-labeled site of CYP2B4, peptide III

(Phe127-Arg140), is located in the C-helix. It is interestingto note that the homologous peptide of CYP101 wasadducted at Met121 by another photoprobe [29]. The pep-

tide IV (Arg434-Arg443), identified as a target of photoreac-tive DIA-N2, belongs to the L-helix. Both C- and L-helicesof CYP2B4 proceed almost parallel underneath the proxi-mal face of the heme plane, creating a hydrophobic groovefor the substrate to enter/escape the active site (see Fig. 9).The distance of backbone carbons (Ca) between both heli-ces �9 A in the CYP2B4 crystal [5] or �6 A in the model[14] spatially allows binding of DIA in the site defined bythe position of Arg133 adducted with the DIA photoaffinityprobe. Thus, peptides III and IV map another outer DIA-binding site of CYP2B4.

DIA modified peptides III and IV of the L- and C-heli-ces, respectively, likely face the cytosol (see Fig. 7).Although the labeling of both peptides was clearly demon-strated, neither a particular substrate access channel isapparent in CYP2B4 structures nor is one predicted bymolecular dynamics simulations at this site. Nevertheless,in this region the DIA saturable binding site was foundby DIA competition experiments. Considering theCYP2B4 plasticity demonstrated for the C-helix [4,5], evenan entry near the access channel invisible in the crystalstructure, might open upon membrane binding and/orredox partner interaction [34]. However, if we take intoaccount the fact that it is not clear whether specificityalways correlates with a functional role for CYP2B4, bind-

ing sites do not necessarily correspond to substrate accesschannels. Such an evaluation might explain the differencesin solutions for the substrate access channel between label-ing studies [present paper] and molecular dynamic simula-tions [7]. Furthermore, the incorporation of the probecould also results from adventitious hydrophobic patcheson the surface of the enzyme that bind DIA, which mightbe another explanation for results observed in the study.

Conclusion

Photoaffinity labeling of CYP2B4 using 3-azidiaman-tane resulted in four labeled peptides. The projection oftheir positions into CYP2B4 structures revealed three bind-ing sites outside the CYP2B4 active site. The first one isdefined by the helices L and C on the proximal heme site,the second one lines with the B–B 0/B 0–C loops and the bstrand 1–4, and the third one is localized between the F/G loop and the pre A-helix region which is likely embeddedin the membrane. These sites mark either routes of entry ofthe putative substrate access channels or possibly serve asthe effector binding sites. Most likely the second and thirdsites define openings to channels 2b/2e and 2f, respectively,which were found by molecular dynamics simulations [7].However, the predominant route 2c (lined by the B/C loop,and the G- and I-helices) expected in the mammalianCYP2C5 [9] and predicted for CYP2B4 [4] was not detectedin our study. Taken together, the present results demon-strate that in the full-length CYP molecules additionalstructure–function relationships exist other than thosefound in crystals of engineered molecules. The significanceof these differences is worth further investigation.

Acknowledgments

Financial support from grants 303/06/0928 and 303/05/2195 from the Grant Agency of Czech Republic, and MSM0021620808 from the Czech Ministry of Education ishighly acknowledged.

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