Table S1. Comparison of activity and affinity of substrates of … · 2011-06-02 · Table S1....

15
Table S1. Comparison of activity and affinity of substrates of human CYP11A1 Compound CYP11A1 human recombinant k cat , min -1 K d , μM Cholesterol 10,7 44.1 Cholestenone 0 weak spectral response a 22R-hydroxycholesterol 47.8 <0.10 20R, 22R-dihydroxycholesterol 148.5 b 10 b 20S-hydroxycholesterol 21.63 4.6 24S-hydroxycholesterol 11.6 10.1 25-hydroxycholesterol 0 weak spectral response 26S-hydroxycholesterol 9.4 23.1 7-dehydrocholesterol (provitamin D 3 ) 12.69 47.6 vitamin D 3 0.018 weak spectral response 25-OH-vitamin D 3 0 weak spectral response Vitamin D 2 0.019 weak spectral response 1α-OH-vitamin D 2 0.032 weak spectral response a The spectral response, ΔA 390-420 or ΔA 414-430 , was below 0.002 absorbance units when 1 μM P450 was titrated with up to 100 μM of compound b k cat and K m for placental human CYP11A1 were determined in (1)

Transcript of Table S1. Comparison of activity and affinity of substrates of … · 2011-06-02 · Table S1....

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Table S1. Comparison of activity and affinity of substrates of human CYP11A1

Compound CYP11A1

human recombinant kcat, min-1 Kd, μM

Cholesterol 10,7 44.1 Cholestenone 0 weak spectral responsea

22R-hydroxycholesterol 47.8 <0.10 20R, 22R-dihydroxycholesterol 148.5b 10b

20S-hydroxycholesterol 21.63 4.6 24S-hydroxycholesterol 11.6 10.1 25-hydroxycholesterol 0 weak spectral response

26S-hydroxycholesterol 9.4 23.1 7-dehydrocholesterol

(provitamin D3) 12.69 47.6

vitamin D3 0.018 weak spectral response 25-OH-vitamin D3 0 weak spectral response

Vitamin D2 0.019 weak spectral response 1α-OH-vitamin D2 0.032 weak spectral response

a The spectral response, ΔA390-420 or ΔA414-430, was below 0.002 absorbance units when 1 μM P450 was titrated with up to 100 μM of compound b kcat and Km for placental human CYP11A1 were determined in (1)

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Table S2. Data collection and refinement statistics.

Sterol Cholesterol PDB: 3N9Y

20SOH-cholesterol PDB: 3NA1

22ROH-cholesterol PDB: 3N9Z

20R,22RdiOH-cholesterol PDB: 3NA0

Data collection Resolution, Å 2.10 2.25 2.17 2.50 Space group P21 P21 P21 P21 Cell dimensions, Å

a,b,c, β

83.0 114.7 86.0 102.0

82.8 115.1 85.7 101.5

83.4 115.1 86.2 101.8

82.8 114.7 85.7 101.8

Molecules in an asymmetric unit 2 2 2 2

Rsym,a, % (last shell) 0.084 (0.522) 0.072 (0.223) 0.112 (0.515) 0.105 (0.365)

I/σ (last shell) 23.5 (3.0) 27.3 (6.4) 15.1 (2.2) 13.0 (2.6) Measured reflections 1025761 876734 1653670 898585 Unique reflections 87091 70161 77768 50846 Redundancy 4.4 4.5 3.8 4.4 Completeness, % (last shell)

99.9 (99.9) 99.3 (98.9) 98.5 (97.4) 98.4 (98.2)

Refinement Rwork

b 0.205 0.193 0.210 0.201 Rfree

c 0.242 0.232 0.243 0.245 No. of residues 1073 985 988 988 No. of waters 509 534 492 291 Wilson plot B-values, Å2 34.7 26.0 26.9 29.3 Ramachandran plot d

Most favored, % (residues)

90.5 (869) 90.2 (788) 90.8 (795) 89.4 (783)

Additional allowed, % (residues)

9.1 (87) 9.3 (81) 8.8 (77) 10.2 (89)

Generously allowed, % (residues)

0.2 (2) 0.3 (3) 0.2 (2) 0.2 (2)

Disallowed, e

% (residues) 0.2 (2) 0.2 (2) 0.2 (2) 0.2 (2)

RMS deviations Bond length, Å 0.010 0.009 0.009 0.010 Bond angle, degrees 1.2 1.2 1.2 1.3

a Rsym=Σhkl [Σi|Ihkl,i − <Ihkl>|]/Σhkl,i <Ihkl>, where Ihkl,i is the intensity of an individual measurement of the reflection with Miller indices h, k, and l, and <Ihkl> is the mean intensity of that reflection. b Rwork=Σ||Fobs|–|Fcalc||/Σ|Fobs|, where |Fobs| and |Fcalc| are observed and calculated structure factor amplitudes, respectively. c Rfree is equivalent to Rwork except that 5% of the total reflections were set aside for an unbiased test of the progress of the refinement. d Program PROCHECK (2) was used. e Residues in disallowed region are in the active site. The disallowed conformation of these residues permits proper ligand binding.

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Table S3. Side-chain cleavage activity and affinity for sterols of fusion proteins.

CYP11A1 Non-fusion enzyme

CYP11A1_Adx fusion (type I linker - 5AAs)

CYP11A1_Adx fusion (type II linker –

23AAs)

CYP11A1_Adx fusion (type III linker – 18AAs)

Compound kcat, min-1 Kd, µM kcat, min-1 Kd, µM kcat, min-1 Kd, µM kcat, min-1 Kd, µM

Cholesterol 10.74± 0.21 44.1 0.047±

0.017 45.7 0.54±0.12 41.4 0.97±0.39 46.8

Cholestenone 0 No

spectral response

0 No

spectral response

0 No

spectral response

0 No

spectral response

7-Dehydrocholesterol (provitamin D3)

12.69± 0.74 47.6 0.083±

0.001 ND 0.49±0.14 ND 0.79± 0.24 ND

22R-hydroxycholesterol

47.81± 2.38

<0.1

0.48± 0.06

<0.1

2.08±0.06

<0.1

2.37± 0.19

<0.1

20S-hydroxycholesterol

21.63± 1.16 4.6 0.121±

0.014 5.2 0.29±0.08 4.4 0.21± 0.01 4.8

Notes: ND – not detected. Type I linker is AAKKT tag usually added to the N-terminus of P450s to improve the expression and solubility in E.coli. Type II linker is from a natural fusion of the cytochrome P450 domain with the ferredoxin domain (CYP51 from Methylococcus capsulatus str). Type III linker is from a natural fusion of the cytochrome P450 domain with the phthalate dioxygenase reductase-like domain (cytochrome P450 RhF from Rhodococcus sp. NCIMB 9784).

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Fig. S1. CYP11A1-catalyzed reactions (indicated as solid black arrows). a, Steroid side-chaincleavage reactions with indications of stable reaction intermediates; b, Hydroxylation ofvitamin D by CYP11A1. Orange arrow indicates site of hydroxylation by CYP11A1(formation of minor products) that occurs after hydroxylation at the C20 carbon of the

OOH

20 2221

asteroid hormones

secosteroid molecule).

HO

O

cholesterol pregnenolone22R-OH H

HO

20

A BC D

Oisocaproic aldehyde

O

HO 7

OHHO7DCHR

7-dehydrocholesterol (provitamin D3) 7-dehydropregnenolone

HO20R,22R-diOH

7-dehydrosteroids(equine type of steroids)

Elevated in humans with Smith-Lemli-Opitzvitamin D3

hv

OHb

Elevated in humans with Smith Lemli Opitz syndrome due to a 7-dehydrocholesterol reductase

(7DCHR) deficiency

20 222317

vitamin D3HO

CH2

20-Hydroxyvitamin D3HO

CH2

OH2317

vitamin D3 20-Hydroxyvitamin D3

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Fig. S2. Interaction of 22R-hydroxycholesterol (yellow) with heme (salmon). The initial MR

difference Fo – Fc map, calculated in the absence of sterol and heme, contoured at 3σ-level and

shown as a blue mesh.

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Fig. S3. a, Superposition of active sites in complexed structures with cholesterol (yellow) and

22R-OH-cholesterol (blue); b, Superposition of active sites in complexed structures with 22R-

OH-cholesterol (blue) and 20,22diOH-cholesterol (green).

a b

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Fig. S4. Access (brown) and egress (orange) channels to/from the active site calculated using CAVER.

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2Fe2S

CYP11A1

Adx

Fig. S5. Superposition of CYP11A1−Adx complex structures with type I and III linkers illustrating the same binding mode within each complex. The CYP11A1 portion is shown as acartoon in either blue or cyan, whereas Adx is shown as sticks with carbons in green or magenta in the respective structures. Oxygen, nitrogen, and sulfur atoms are in red, blue, and yellow,respectively. 2Fe2S cluster is shown as spheres and labeled. The linkers are not visible in the crystalline state.

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Fig. S6. Multiple sequence alignment of mitochondrial cytochrome P450 proteins of subphylum Vertebrata. Numbers on top are shown for the mature form of human CYP11A1. A heme-bound cysteine is indicated by an asterisk. Blue and green lines indicate residues involved in the interaction of bovine CYP11A1 with Adx based on chemical modifications (3-5) and site-directed mutagenesis data (6-9), respectively. The residues in the interaction interface are indicated by triangles, with red triangles highlighting the residues making hydrogen bonds and salt bridges with Adx. hum- Homo sapiens; bov - Bos taurus; pig - Sus scrofa; rat - Rattus norvegicus; dog- Canis familiaris; gal - Gallus gallus; ran - Rana catesbeiana; dan - Danio rerio. 11A1_hum – mature form of human CYP11A1; 11A1phum – precursor of human CYP11A1 (AAA52162); 11A1_bov - (AAI33390); 11A1_rat - (NP_058982); 11A1_dog - (XP_535539); 11A1_pig - (NP_999592); 11A1_gal - (NP_001001756); 11A1_ran - (ACL36104); 11A1_dan - (NP_694485); 11B1_hum - (NP_000488); 11B2_hum - (NP_000489); 11B1_bov - (P15150); 11B1_rat - (NP_036669); 11B2_rat - (NP_036670); 11B3_rat - (NP_861545); 27A1_hum - (NP_000775); 27A1_bov - (AAI23879); 27A1_rat - (NP_849178); 27B1_hum - (NP_000776); 27B1_bov - (XP_588481); 27B1_rat - (NP_446215); 24A1_rat - (NP_963966); 24A1_hum - (NP_000773).

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10 20 30 40 11A1_hum: ----------------------------------------------I--S-TRSPR---PFNEIPSPGDNGWLN-LYHFWRETGTHKVHLHHV : 40 11A1phum: -------MLAKGLPPRSVLVKGYQTFLSAPREGLGRLRVPTGEGAGI--S-TRSPR---PFNEIPSPGDNGWLN-LYHFWRETGTHKVHLHHV : 79 11A1_bov: -------MLARGLPLRSALVKACPPILSTVGEGWGHHRVGTGEGAGI--S-TKTPR---PYSEIPSPGDNGWLN-LYHFWREKGSQRIHFRHI : 79 11A1 rat: -------MLAKGLCLRSVLVKSCQPFLSPVWQGPG---LATGNGAGI--SSTNSPR---SFNEIPSPGDNGWIN-LYHFLRENGTHRIHYHHM : 77

´αAععععععععععععع αAعععععععع

_ Q Q11A1_dog: -------MLAKGLPLRSVLVKGCQPFLSTVWEGPGHPRVPTGDGASI--S-TQIPR---PFSEIPTPGNNGWLN-LYNFWREMGSQKIHYHQV : 79 11A1_pig: -------MLARGLALRSVLVKGCQPFLSAPRECPGHPRVGTGEGACI--S-TKTPR---PFSEIPSPGDNGWIN-LYRFWKEKGTQKIHYHHV : 79 11A1_gal: ------------------MLSRAAPIAGSFQACRCAGGIPALAGVHYPLPSSSGAR---PFDQVPGEWRAGWLN-LYHFWKEGGFHNVHNIMA : 71 11A1_ran: -----------------MMLSRRLCLLPSSSGMLNYHLVVSESSSMIHNQSGTSPL---PYDQLPGDWRNGWSG-LYHFFRKDGFHNIHHLME : 72 11A1_dan: ------------MARWNVTFAR---LDQSLSSLKNLLQVKVTRSGRAPQNSTVQ-----PFNKIPGRWRNSLLS-VLAFTKMGGLRNVHRIMV : 72 11B1_hum: -----------------------MALRAKAEVCMAVPWLSLQRAQALGTRAARVPRTVLPFEAMPRRPGNRWLR-LLQIWREQGYEDLHLEVH : 69 11B2_hum: -----------------------MALRAKAEVCVAAPWLSLQRARALGTRAARAPRTVLPFEAMPQHPGNRWLR-LLQIWREQGYEHLHLEMH : 69 11B1_bov: -----------------------MALWAKARVRMAGPWLSLHEARLLGTRGAAAPKAVLPFEAMPRCPGNKWMR-MLQIWKEQSSENMHLDMH : 69 11B1_rat: -----------------------MALRVTADVWLARPWQCLHRTRALGTTAKVAPKTLKPFEAIPQYSRNKWLK-MIQILREQGQENLHLEMH : 69 11B2_rat: -----------------------MALRVTADVWLARPWQCLHRTRALGTTATLAPKTLKPFEAIPQYSRNKWLK-MIQILREQGQENLHLEMH : 69 11B3_rat: -----------------------MALRVTADVW-ARPWQCLHRTRALGSTATQAPKTLKPFEAIPQYSRNKWLK-MIQILREQSQENLHLEMH : 68 27A1_hum: MAA-LGCARLRWAL---RGAGRGLCPHGARAKAAIPAALPSDKATGAPGAGPGVRRRQRSLEEIPRLGQ---LRFFFQLFVQGYALQLHQLQV : 8627A1_bov: MGA-LGSARLRWALLGRRAALPGLGSFGARAKAAIPSALPAAQAAEAPGTGPG-DRRLRSLDELSGPGQ---LRLLFQLLVQGYVLHLHQLQV : 88 27A1_rat: MAV-LSRMRLRWALLDTRVMGHGLCPQGARAKAAIPAALRDHESTEGPGTGQD-RPRLRSLAELPGPGT---LRFLFQLFLRGYVLHLHELQA : 88 27B1_hum: ------------------------MTQTLKYASRVFHRVRWAPELGASLGYREYHSARRSLADIPGPST---PSFLAELFCKGGLSRLHELQV : 66 27B1_bov: ------------------------MTQTLKFASRVFHRVR-CPELGASLGSRGSESAPRVLADIPGPST---PGFLAELFCKGGLSRLHELQV : 65 27B1_rat: ------------------------MTQAVKLASRVFHRV----QLPSQLGS---DSVLRSLSDIPGPST---PSFLAELFCKGGLSRLHELQV : 59 24A1_rat: MSCPIDKRRTLIAFL-RRLRDLGQPPRSVTSKASASRAPKEVPLCPLMTDGE-----TRNVTSLPGPTNWPLLGSLLEIFWKGGLKKQHDTLA : 87 24A1_hum: MSSPISKSRSLAAFL-QQLRSPRQPPRLVTSTAYTSPQPREVPVCPLTAGGE-----TQNAAALPGPTSWPLLGSLLQILWKGGLKKQHDTLV : 87

β1‐1 β1‐2αAععععععع عععععععععع عععععععععععع ععععععععععععععععع αB´αBععععععععععععع αC αDαC´

50 60 80 90 100 130 11A1_hum: QNFQKYGPIYREKLGNVESVYVIDPEDVALLFKSEGPNPERFLIPPWVAYHQYYQRPIGVLLKKSAAWKKDRVALNQEVMAPEATKNFLPLLD : 133 11A1phum: QNFQKYGPIYREKLGNVESVYVIDPEDVALLFKSEGPNPERFLIPPWVAYHQYYQRPIGVLLKKSAAWKKDRVALNQEVMAPEATKNFLPLLD : 172 11A1_bov: ENFQKYGPIYREKLGNLESVYIIHPEDVAHLFKFEGSYPERYDIPPWLAYHRYYQKPIGVLFKKSGTWKKDRVVLNTEVMAPEAIKNFIPLLN : 172 11A1_rat: QNFQKYGPIYREKLGNMESVYILDPKDAATLFSCEGPNPERYLVPPWVAYHQYYQRPIGVLFKSSDAWRKDRIVLNQEVMAPDSIKNFVPLLE : 170 11A1_dog: QNFQKYGPIYREKLGSVESVYIIDPEDVALLFKFEGPTPERFCIPPWVAYHQYHQRPVGVLLKKSGAWKKDRLALNQEVMAPEAIKNFIPLLD : 172 11A1_pig: QNFQKYGPIYREKLGNLESVYIIDPEDVALLFKFEGPNPERYNIPPWVAYHQHYQKPVGVLLKKSGAWKKDRLVLNTEVMAPEAIKNFIPLLD : 172 11A1_gal: SKFQRFGPIYREKLGVYESVNIISPRDAATLFKSEGMLPERFSVPPWVAYRDYRNKPYGVLLKTGEAWRSDRLTLNKEVLSPQVVDSFVPLLD : 164 11A1_ran: ENYQRFGPIYREKLGTYDSVYIQRPEDAAILFQVEGIHPERLRIQPWFEYRDYRNKKYGVLLKSGEDWRCQRLTLNREVLSVAGMNRFLPLLD : 165 11A1_dan: HNFKTFGPIYREKVGIYDSVYIIKPEDGAILFKAEGHHPNRINVDAWTAYRDYRNQKYGVLLKEGKAWKTDRMILNKELLLPKLQGTFVPLLE : 165 11B1 hum: QTFQELGPIFRYDLGGAGMVCVMLPEDVEKLQQVDSLHPHRMSLEPWVAYRQHRGHKCGVFLLNGPEWRFNRLRLNPEVLSPNAVQRFLPMVD : 16211B1_hum: QTFQELGPIFRYDLGGAGMVCVMLPEDVEKLQQVDSLHPHRMSLEPWVAYRQHRGHKCGVFLLNGPEWRFNRLRLNPEVLSPNAVQRFLPMVD : 16211B2_hum: QTFQELGPIFRYNLGGPRMVCVMLPEDVEKLQQVDSLHPCRMILEPWVAYRQHRGHKCGVFLLNGPEWRFNRLRLNPDVLSPKAVQRFLPMVD : 162 11B1_bov: QTFQELGPIFRYDVGGRHMVFVMLPEDVERLQQADSHHPQRMILEPWLAYRQARGHKCGVFLLNGPQWRLDRLRLNPDVLSLPALQKYTPLVD : 162 11B1_rat: QAFQELGPIFRHSAGGAQIVSVMLPEDAEKLHQVESILPHRMPLEPWVAHRELRGLRRGVFLLNGADWRFNRLQLNPNMLSPKAIQSFVPFVD : 162 11B2_rat: QAFQELGPIFRHSAGGAQIVSVMLPEDAEKLHQVESILPRRMHLEPWVAHRELRGLRRGVFLLNGAEWRFNRLKLNPNVLSPKAVQNFVPMVD : 162 11B3_rat: QAFQELGPIFRHSAGGAQIVSVMLPEDAEKLHQVESILPRRMTLESWVAHRELRGLRRGVFLLNGADWRFNRLQLNPNMLSPKAVQSFVPFVD : 161 27A1_hum: LYKAKYGPMWMSYLGPQMHVNLASAPLLEQVMRQEGKYPVRNDMELWKEHRDQHDLTYGPFTTEGHHWYQLRQALNQRLLKPAEAALYTDAFN : 179 27A1_bov: LNKAKYGPIWINRVGPQMHVHLASAPLLEQVMRQEGKYPVRDDMKLWKEHRDQQGLSYGPFTTMGEQWYRLRQTLNQRMLKPAEAALYTDALN : 181 27A1_rat: LNKAKYGPMWTTTFGTRTNVNLASAPLLEQVMRQEGKYPIRDSMEQWKEHRDHKGLSYGIFITQGQQWYHLRHSLNQRIVKPAEAALYTDALN : 181 27B1_hum: QGAAHFGPVWLASFGTVRTVYVAAPALVEELLRQEGPRPERCSFSPWTEHRRCRQRACGLLTAEGEEWQRLRSLLAPLLLRPQAAARYAGTLN : 159 27B1_bov: QGAARFGPVWLASFGTVRTVYLAAPTLVEQLLRQEGPRPERCSFSPWTEHRRRRQRACGLLTAEGEEWQRLRSLLAPLLLRPQAAARYAGTLH : 158 27B1 rat: HGAARYGPIWSGSFGTLRTVYVADPALVEQLLRQESHCPERCSFSSWSEHRRRHQRACGLLTADGEEWQRLRSLLAPLLLRPQAAAGYAGTLD : 152_24A1_rat: EYHKKYGQIFRMKLGSFDSVHLGSPSLLEALYRTESAHPQRLEIKPWKAYRDHRNEAYGLMILEGQEWQRVRSAFQKKLMKPVEIMKLDKKIN : 180 24A1_hum: EYHKKYGKIFRMKLGSFESVHLGSPCLLEALYRTESAYPQRLEIKPWKAYRDYRKEGYGLLILEGEDWQRVRSAFQKKLMKPGEVMKLDNKIN : 180 140 160 170 180 190 200 210 220 11A1_hum: AVSRDFVSVLHRRIKKAGSG-NYSGDISDDLFRFAFESITNVIFGERQGMLEEVVNPEAQRFIDAIYQMFHTSVPMLNLPPDLFRLFRTKTWK : 225 11A1phum: AVSRDFVSVLHRRIKKAGSG-NYSGDISDDLFRFAFESITNVIFGERQGMLEEVVNPEAQRFIDAIYQMFHTSVPMLNLPPDLFRLFRTKTWK : 264 11A1_bov: PVSQDFVSLLHKRIKQQGSG-KFVGDIKEDLFHFAFESITNVMFGERLGMLEETVNPEAQKFIDAVYKMFHTSVPLLNVPPELYRLFRTKTWR : 264 11A1_rat: GVAQDFIKVLHRRIKQQNSG-KFSGDISDDLFRFAFESITSVVFGERLGMLEEIVDPESQRFIDAVYQMFHTSVPMLNMPPDLFRLFRTKTWK : 262

ععععععععععععععععع ععععععععععععععععع عععععععععععععععععع ععععععععععععععαF´αD β3-1 αFαE αG

11A1_dog: PVSQDFVKVLHRRIKQQGSG-KFSGDISDDLFRFAFESITNVMFGERLGMLEERVDPEAQRFIDAVYQMFHTSVPMLTFPPDLFRLFKTKTWR : 26411A1_pig: TVSQDFVGVLHRRIKQQGSG-KFSGDIREDLFRFAFESITNVIFGERLGMLEEIVDPEAQKFIDAVYQMFHTSVPMLNLPPDLFRLFRTKTWR : 264 11A1_gal: QVSQDFLRRARAQVQQSGRE-RWTADFSHELFRFALESVCHVLYGERLGLLQDFVDPEAQQFIDAVTLMFHTTSPMLYVPPALLRHLNTKTWR : 256 11A1_ran: SVGQDFVRRVYTHVERSGRG-KWTADLSQELFRFALESVCNVLYGQRLGLLQDYINPESQEFIDSINLMFDTTSPMLYIPPRVFRLMNLSVWK : 257 11A1_dan: EVGQDFVARVNKQIERSGQK-QWTTDLTHDLFKFSLESVSAVLYGERLGLLLDNIDPEFQHFIDCVSVMFKTTSPMLYLPPGLLRSIGSNIWK : 257 11B1_hum: AVARDFSQALKKKVLQNARG-SLTLDVQPSIFHYTIEASNLALFGERLGLVGHSPSSASLNFLHALEVMFKSTVQLMFMPRSLSRWTSPKVWK : 254 11B2_hum: AVARDFSQALKKKVLQNARG-SLTLDVQPSIFHYTIEASNLALFGERLGLVGHSPSSASLNFLHALEVMFKSTVQLMFMPRSLSRWISPKVWK : 254 11B1_bov: GVARDFSQTLKARVLQNARG-SLTLDIAPSVFRYTIEASTLVLYGERLGLLTQQPNPDSLNFIHALEAMLKSTVQLMFVPRRLSRWMSTNMWR : 254 11B1_rat: VVARDFVENLKKRMLENVHG-SMSINIQSNMFNYTMEASHFVISGERLGLTGHDLKPESVTFTHALHSMFKSTTQLMFLPKSLTRWTSTRVWK : 254 11B2_rat: EVARDFLEALKKKVRQNARG-SLTMDVQQSLFNYTIEASNFALFGERLGLLGHDLNPGSLKFIHALHSMFKSTTQLLFLPRSLTRWTSTQVWK : 254 11B3_rat: VVARDFVENLKKRMLENVHG-SMSMDIQSNVFNYTMEASHFVISGERLGLTGHDLNPESLKFIHALHSMFKSTTQLMFLPKNLTRWTSTQVWK : 253 27A1 hum: EVIDDFMTRLDQLRAESASGNQ VSDMAQLFYYFALEAICYILFEKRIGCLQRSIPEDTVTFVRSIGLMFQNSLYATFLPKWTRPVLPF WK : 26927A1_hum: EVIDDFMTRLDQLRAESASGNQ-VSDMAQLFYYFALEAICYILFEKRIGCLQRSIPEDTVTFVRSIGLMFQNSLYATFLPKWTRPVLPF--WK : 26927A1_bov: EVINDFMDQLKQLRAESASGDH-VPDIAHQFYFFALEAISYILFEKRIGCLERSIPKDTETFVRSVGLMFHNSLFVTFLPTWTRPLLPF--WK : 271 27A1_rat: EVISDFIARLDQVRTESASGDQ-VPDVANLLYHLALEAICYILFEKRVGCLEPSIPEDTATFIRSVGLMFKNSVYVTFLPKWSRPLLPF--WK : 271 27B1_hum: NVVCDLVRRLRRQRGRGTGPPALVRDVAGEFYKFGLEGIAAVLLGSRLGCLEAQVPPDTETFIRAVGSVFVSTLLTMAMPHWLRHLVPGP-WG : 251 27B1_bov: GVVRDLVRRLRRQRGLGAGPPSLVRDVAGEFYKFGLEGIAAVLLGSRLGCLEAEVPPDTETFIRAVGSVFVSTLLTMAMPSWLHRVVPGP-WD : 250 27B1_rat: SVVSDLVRRLRRQRGRGSGLPDLVLDVAGEFYKFGLEGIGAVLLGSRLGCLEAEVPPDTETFIEAVGSVFVSTLLTMAMPSWLHRLIPGP-WA : 244 24A1_rat: EVLADFLERMDELCDERGR----IPDLYSELNKWSFESICLVLYEKRFGLLQKETEEEALTFITAIKTMMSTFGKMMVTPVELHKRLNTKVWQ : 269 24A1_hum: EVLADFMGRIDELCDERGH----VEDLYSELNKWSFESICLVLYEKRFGLLQKNAGDEAVNFIMAIKTMMSTFGRMMVTPVELHKSLNTKVWQ : 269

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230 240 250 260 280 290 300 310 11A1_hum: DHVAAWDVIFSKADIYTQNFYWELRQKGSVHHDYRGM-----LYRLLGDSKMSFEDIKANVTEMLAGGVDTTSMTLQWHLYEMARNLKVQDML : 313 11A1phum: DHVAAWDVIFSKADIYTQNFYWELRQKGSVHHDYRGM-----LYRLLGDSKMSFEDIKANVTEMLAGGVDTTSMTLQWHLYEMARNLKVQDML : 352 11A1_bov: DHVAAWDTIFNKAEKYTEIFYQDLRRKTEFR-NYPGI-----LYCLLKSEKMLLEDVKANITEMLAGGVDTTSMTLQWHLYEMARSLNVQEML : 351 11A1_rat: DHAAAWDVIFSKADEYTQNFYWDLRQKRDFS-KYPGV-----LYSLLGGNKLPFKNIQANITEMLAGGVDTTSMTLQWNLYEMAHNLKVQEML : 349 11A1_dog: DHVAAWDVIFNKAEIYTQNFYWELRQKRDVD-NYPGI-----LHRLLKSNKLLFEDVKANITEMLAGGVDTTSMTLQWHLYEMARSLEVQEVL : 351 11A1 pig: DHVAAWDTIFNKAEKYTQNFYWDLRRKREFN-NYPGI-----LYRLLGNDKLLSEDVKANVTEMLAGGVDTTSMTLQWHLYEMARSLNVQEML : 351

عععععععععععععععععععععععععععععععععععععععععععععععععع ععععععع عععععععععععععععععععععععععععععععععععععععععععععععععع ععععععععαH αIαG αJ

_p g Q Q Q11A1_gal: DHVHAWDAIFTQADKCIQNVYRDIRLQRKSTEEHTGI-----LFSLLVQDKLPLDDIKASVTEMMAGGVDTTSMTLQWAMLELARSPGIQERL : 344 11A1_ran: NHVKSWDAIFNHADLCIQGIYSSLR-QR-SDNTYSGV-----LSSLLLQHQLPLEDIKASITELMAGGVDTTSMTLQWAMYELARAPSVQEKL : 343 11A1_dan: NHVEAWDGIFNQADRYIQNIFKQWKENPEGNGKYPGV-----LAILLMQDKLSIEDIKASVTELMAGGVDSVTFTLLWTLYELARQPDLQDEL : 345 11B1_hum: EHFEAWDCIFQYGDNCIQKIYQELAFSRPQQ--YTSI-----VAELLLNAELSPDAIKANSMELTAGSVDTTVFPLLMTLFELARNPNVQQAL : 340 11B2_hum: EHFEAWDCIFQYGDNCIQKIYQELAFNRPQH--YTGI-----VAELLLKAELSLEAIKANSMELTAGSVDTTAFPLLMTLFELARNPDVQQIL : 340 11B1_bov: EHFEAWDYIFQYANRAIQRIYQELALGHPWH--YSGI-----VAELLMRADMTLDTIKANTIDLTAGSVDTTAFPLLMTLFELARNPEVQQAV : 340 11B1_rat: EHFDSWDIISEYVTKCIKNVYRELAEGRQQS--WS-V-----ISEMVAQSTLSMDAIHANSMELIAGSVDTTAISLVMTLFELARNPDVQQAL : 339 11B2_rat: EHFDAWDVISEYANRCIWKVHQELRLGSSQT--YSGI-----VAALITQGALPLDAIKANSMELTAGSVDTTAIPLVMTLFELARNPDVQQAL : 340 11B3_rat: GHFESWDIISEYVTKCIKNVYRELAEGRQQS--WS-V-----ISEMVAQSTLSMDAIHANSMELIAGSVDTTAISLVMTLFELARNPDVQQAL : 338 27A1_hum: RYLDGWNAIFSFGKKLIDEKLEDMEAQLQAAGPDGIQVSG-YLHFLLASGQLSPREAMGSLPELLMAGVDTTSNTLTWALYHLSKDPEIQEAL : 361 27A1_bov: RYLDGWNTIFSFGKKLIDQKLEEIEAQLKTENPEKTQISG-YLHFLLTSGQLSPREAEGSLPELLLAGVDTTSNTLTWALYHLSKNPEIQAAL : 363 27A1_rat: RYMNNWDNIFSFGEKMIHQKVQEIEAQLQAAGPDGVQVSG-YLHFLLTKELLSPQETVGTFPELILAGVDTTSNTLTWALYHLSKNPEIQEAL : 36327B1_hum: RLCRDWDQMFAFAQRHVERREAEAAMRNGGQ-PEKDLESGAHLTHFLFREELPAQSILGNVTELLLAGVDTVSNTLSWALYELSRHPEVQTAL : 343 27B1_bov: RLCRDWDQMFAFAQQHVEQREAEVAMRNQSEKSEEDMGPGAHLTYFLLQKELPAASILGNVTELLLAGVDTVSNTLSWALYELSRHPEIQTAL : 343 27B1_rat: RLCRDWDQMFAFAQKHVEQREGEAAVRNQGK-PEEDLPTGHHLTHFLFREKVSVQSIVGNVTELLLAGVDTVSNTLSWALYELSRHPEVQSAL : 336 24A1_rat: AHTLAWDTIFKSVKPCIDNRLQRYSQQ-----PGAD-----FLCDIYQQDHLSKKELYAAVTELQLAAVETTANSLMWILYNLSRNPQAQRRL : 352 24A1_hum: DHTLAWDTIFKSVKACIDNRLEKYSQQ-----PSAD-----FLCDIYHQNRLSKKELYAAVTELQLAAVETTANSLMWILYNLSRNPQVQQKL : 352 320 330 350 360 370 380 390 400 11A1 h RAEVLAA RHQAQGDMATMLQLVPLLKASIKETLRLHPISVTLQRYL VNDLVLRDYMIPAKTLVQVAIYALGREPTFFFDPENFDPTRWLS 403

عععععععععععع عععع ععععععععععععع عععععع αKعععع αK´β1-3β1-4 β2-2β2-1αJ´αJ αK´´

11A1_hum: RAEVLAA--RHQAQGDMATMLQLVPLLKASIKETLRLHPISVTLQRYL-VNDLVLRDYMIPAKTLVQVAIYALGREPTFFFDPENFDPTRWLS : 40311A1phum: RAEVLAA--RHQAQGDMATMLQLVPLLKASIKETLRLHPISVTLQRYL-VNDLVLRDYMIPAKTLVQVAIYALGREPTFFFDPENFDPTRWLS : 442 11A1_bov: REEVLNA--RRQAEGDISKMLQMVPLLKASIKETLRLHPISVTLQRYP-ESDLVLQDYLIPAKTLVQVAIYAMGRDPAFFSSPDKFDPTRWLS : 441 11A1_rat: RAEVLAA--RRQAQGDMAKMVQLVPLLKASIKETLRLHPISVTLQRYI-VNDLVLRNYKIPAKTLVQVASYAMGRESSFFPNPNKFDPTRWLE : 439 11A1_dog: REEVLAA--RRQAQGNVSTMLQLVPLLKASIKETLRLHPISVTLQRYL-ENDLVLRNYMIPAKTLVQVSTYAMGQDPTFFLNPSKFDPTRWLG : 441 11A1_pig: REEVLNA--RRQAQGDTSKMLQLVPLLKASIKETLRLHPISVTLQRYL-VNDLVLRDYMIPAKTLVQVAVYAMGRDPAFFSNPGQFDPTRWLG : 441 11A1_gal: RAEVLAA--KQEAQGDRVKMLKSIRLLKAAIKETLRLHPVAVTLQRYT-TQEVILQDYRIPPKTLVQVGLYAMGRDPEVFPKPEQFNPERWLV : 434 11A1_ran: RSEIKAA--RKAAGNDLNALLKRIPLVKAALKETLRLHPVAISLQRYT-QRDTVIRNYMIPRGTLVQVGLYAMGRNPDIFPSPEKFFPERWLG : 433 11A1_dan: RAEISAA--RIAFKGDMVQMVKMIPLLKAALKETLRLHPVAMSLPRFI-TEDTVIQNYHIPAGTLVQLGVYAMGRDHQFFPKPEQYCPSRWIS : 435 11B1_hum: RQESLAA--AASISEHPQKATTELPLLRAALKETLRLYPVGLFLERVA-SSDLVLQNYHIPAGTLVRVFLYSLGRNPALFPRPERYNPQRWLD : 430 11B2_hum: RQESLAA--AASISEHPQKATTELPLLRAALKETLRLYPVGLFLERVV-SSDLVLQNYHIPAGTLVQVFLYSLGRNAALFPRPERYNPQRWLD : 430 11B1 bov: RQESLVA--EARISENPQRAITELPLLRAALKETLRLYPVGITLEREV-SSDLVLQNYHIPAGTLVKVLLYSLGRNPAVFARPESYHPQRWLD : 43011B1_bov: RQESLVA EARISENPQRAITELPLLRAALKETLRLYPVGITLEREV SSDLVLQNYHIPAGTLVKVLLYSLGRNPAVFARPESYHPQRWLD : 43011B1_rat: RQESLAA--EASIVANPQKAMSDLPLLRAALKETLRLYPVGSFVERIV-HSDLVLQNYHVPAGTFVIIYLYSMGRNPAVFPRPERYMPQRWLE : 429 11B2_rat: RQETLAA--EASIAANPQKAMSDLPLLRAALKETLRLYPVGGFLERIL-NSDLVLQNYHVPAGTLVLLYLYSMGRNPAVFPRPERYMPQRWLE : 430 11B3_rat: RQESLAA--EASIAANPQKAMSDLPLLRAALKETLRLYPIGSSLERIV-DSDLVLQNYHVPAGTLVIIYLYSMGRNPAVFPRPERYMPQRWLE : 428 27A1_hum: HEEVVGVVPAGQV--PQHKDFAHMPLLKAVLKETLRLYPVVPTNSRII-EKEIEVDGFLFPKNTQFVFCHYVVSRDPTAFSEPESFQPHRWLR : 451 27A1_bov: HKEVVGVVPAGQV--PQHKDLARMPLLKAVLKETLRLYPVVPVNSRVVVDKEIEVGGFLFPKNTQFVLCHYVISRDPDIYPEPDSFQPQRWLR : 454 27A1_rat: HKEVTGVVPFGKV--PQNKDFAHMPLLKAVIKETLRLYPVVPTNSRIITEKETEINGFLFPKNTQFVLCHYVVSRDPSVFPEPESFQPHRWLR : 454 27B1_hum: HSEITAALSPGSSAYPSATVLSQLPLLKAVVKEVLRLYPVVPGNSRV-PDKDIHVGDYIIPKNTLVTLCHYATSRDPAQFPEPNSFRPARWL- : 434 27B1_bov: HAEITAALGPGSSTQPSATALSQLPLLKAVVKEVLRLYPVVPGNSRV-PDRDICVGEYIIPKNTLVTLCHYATSRDPAQFPEPNSFRPARWL- : 434 27B1_rat: HSEITGAVNPGSYAHLQATALSQLPLLKAVIKEVLRLYPVVPGNSRV-PDRDICVGNYVIPQDTLVSLCHYATSRDPAQFREPNSFNPARWL- : 427 24A1_rat: LQEVQSVLPDNQT--PRAEDLRNMPYLKACLKESMRLTPSVPFTTRTL-DKPTVLGEYALPKGTVLTLNTQVLGSSEDNFEDSHKFRPERWLQ : 442 24A1 hum: LKEIQSVLPENQV--PRAEDLRNMPYLKACLKESMRLTPSVPFTTRTL-DKATVLGEYALPKGTVLMLNTQVLGSSEDNFEDSSQFRPERWLQ : 442_ Q Q Q Q Q 410 420 440 450 460 470 480 11A1_hum: KDKNITY-----FRNLGFGWGVRQCLGRRIAELEMTIFLINMLENFRVEIQ-HLSDVGTTFNLILMPEKPISFTFWPFNQE-----ATQQ--- : 482 11A1phum: KDKNITY-----FRNLGFGWGVRQCLGRRIAELEMTIFLINMLENFRVEIQ-HLSDVGTTFNLILMPEKPISFTFWPFNQE-----ATQQ--- : 521 11A1_bov: KDKDLIH-----FRNLGFGWGVRQCVGRRIAELEMTLFLIHILENFKVEMQ-HIGDVDTIFNLILTPDKPIFLVFRPFNQD-----PPQA--- : 520 11A1_rat: KSQNTTH-----FRYLGFGWGVRQCLGRRIAELEMTIFLINVLENFRIEVQ-SIRDVGTKFNLILMPEKPIFFNFQPLKQDLGSTMPRKGDTV : 526 11A1_dog: KDKELIH-----FRNLGFGWGVRQCVGRRIAELEMTLFLIHILENFRVEMQ-QLRDVGTTFNLILMPDKPIFLTFRPFNQG-----PPQV--- : 520 11A1_pig: KERDLIH-----FRNLGFGWGVRQCVGRRIAELEMTLFLIHILENFKVELQ-HFSDVDTIFNLILMPDKPIFLVFRPFNQD-----PLQA--- : 520

عععععععععععععععععععαL β3−3 β3-2β4-2β4-1meander

*

11A1_gal: MGSK--H-----FKGLSFGFGPRQCLGRRIAELEMQLFLMHILENFKIETK-RAVEVGTKFDLILVPEKPIYLRLRPLQPQE----------- : 50811A1_ran: GEST--H-----FRSLGFGFGPRQCLGRRIAEMEMNLFLIHILEHFRIETN-RMIEVGTTFKLILFPFKPIHLTLRPLDD------------- : 505 11A1_dan: SKRQ--Y-----FKSLGFGFGPRQCLGRRIAETEMQIFLIHMLENFRIEKQ-KQIEVRSKFELLLMPEKPIMLTIKPLNASR----------- : 509 11B1_hum: IRGSGRN-----FYHVPFGFGMRQCLGRRLAEAEMLLLLHHVLKHLQVETL-TQEDIKMVYSFILRPSMFPLLTFRAIN-------------- : 503 11B2_hum: IRGSGRN-----FHHVPFGFGMRQCLGRRLAEAEMLLLLHHVLKHFLVETL-TQEDIKMVYSFILRPGTSPLLTFRAIN-------------- : 503 11B1_bov: RQGSGSR-----FPHLAFGFGVRQCLGRRVAEVEMLLLLHHVLKNFLVETL-EQEDIKMVYRFILMPSTLPLFTFRAIQ-------------- : 503 11B1_rat: RK---RS-----FQHLAFGFGVRQCLGRRLAEVEMLLLLHHMLKTFQVETL-RQEDMQMVFRFLLMPSSSPFLTFRPVS-------------- : 499 11B2_rat: RK---RS-----FQHLAFGFGVRQCLGRRLAEVEMLLLLHHMLKTFQVETL-RQEDVQMAYRFVLMPSSSPVLTFRPIS-------------- : 500 11B3_rat: RK---RS-----FQHLAFGFGVRQCLGRRLAEVEVLLLLHHMLKIFQVETL-RQEDVQMAYRFVLMPNPRLVLTIRPVS-------------- : 498 27A1_hum: NSQPATPRIQHPFGSVPFGYGVRACLGRRIAELEMQLLLARLIQKYKVVLAPETGELKSVARIVLVPNKKVGLQFLQRQC------------- : 531 27A1_bov: KNQPDALKTQHPFGSVPFGYGVRACLGRRIAELEMQLLLTRLIQHYEVVLAPETGEVTSVARIVLVPNKKVGLRFLQRQS------------- : 534 27A1 t KREDDNSGIQHPFGSVPFGYGVRSCLGRRIAELEMQLLLSRLIQKYEVVLSPGMGEVKSVSRIVLVPSKKVSLRFLQRQ 53327A1_rat: KREDDNSGIQHPFGSVPFGYGVRSCLGRRIAELEMQLLLSRLIQKYEVVLSPGMGEVKSVSRIVLVPSKKVSLRFLQRQ-------------- : 53327B1_hum: -GEGPTP---HPFASLPFGFGKRSCMGRRLAELELQMALAQILTHFEVQPEPGAAPVRPKTRTVLVPERSINLQFLDR--------------- : 508 27B1_bov: -GEGPAP---HPFASLPFGFGKRSCVGRRLAELELQMALAQILIHFEVQPEPGSAPVRPMTRTVLVPERSINLQFVDR--------------- : 508 27B1_rat: -GEGPAP---HPFASLPFGFGKRSCIGRRLAELELQMALAQILTHFEVLPEPGALPVKPMTRTVLVPERSIHLQFVDR--------------- : 501 24A1_rat: KEKKI-----NPFAHLPFGIGKRMCIGRRLAELQLHLALCWIIQKYDIVATDNE-PVEMLHLGILVPSRELPIAFRPR--------------- : 514 24A1_hum: EKEKI-----NPFAHLPFGVGKRMCIGRRLAELQLHLALCWIVRKYDIQATDNE-PVEMLHSGTLVPSRELPIAFCQR--------------- : 514 C-GRR--E

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Fig. S7. Molecular surface representation of the binding site on Adx with a, CYP11A1 (green; the CYP11A1-Adx interface is approximately 1040 Å2 of the surface area), b, AdR (red) based on the structure of the complex AdR-Adx, PDB: 1E6E. The AdR-Adx interface is approximately 1166 Å2 of the surface area. c, Overlay of the interaction sites of Adx for both redox-partners (orange). Light grey in panel a indicates distal disordered regions of Adx, which are shown the same as in b. Residues of Adx forming salt bridges with AdR and CYP11A1 are indicated.

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Fig. S8. Reduction of Adx-CYP11A1fusions monitored by CO-difference spectra. For chemical reduction, sodium dithionite was used, and for enzymatic reduction, proteins and NADPH were added as described in the Methods.

0,0

10,0

20,0

30,0

40,0

50,0

60,0

CYP11A1_Adx fusion (type I linker - 5AAs)

CYP11A1_Adx fusion (type II linker - 23AAs)

CYP11A1_Adx fusion (type III linker - 18AAs)

CYP11A1

% to

che

mic

al r

educ

tion

ADRADR+cholADR+ADXADR+ADX+chol

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Cys52

2Fe2S

Cys55

Cys92

Cys46a

b

Lys343

Lys339

Asp72

Asp76Asp79Glu73

Glu74

Fig. S9. a, Anomalous difference Fourier map (red) of the 2Fe2S-cluster contoured at 4σ. Thedata was collected at 1.0 Å wavelength. b, Simulated annealing 2Fo – Fc map contoured at 1σ for residues of the interaction helix of Adx (gray) and the K-helix of CYP11A1 (green) showing the two salt bridges (see text for details). This view corresponds to that shown in panel a with 90° clockwise rotation along the vertical axis.

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REFERENCES 1. Tuckey RC, Cameron KJ (1993) Side-chain specificities of human and bovine

cytochromes P-450scc. Eur J Biochem 217:209-215. 2. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK - a

program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291.

3. Adamovich TB, Pikuleva IA, Chashchin VL, Usanov SA (1989) Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin. Biochim Biophys Acta 996:247-253.

4. Tsubaki M, Iwamoto Y, Hiwatashi A, Ichikawa Y (1989) Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal 5'-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc. Biochemistry 28:6899-6907.

5. Tuls J, Geren L, Millett F (1989) Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding. J Biol Chem 264:16421-16425.

6. Wada A, Waterman MR (1992) Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding. J Biol Chem 267:22877-22882

7. Strushkevich NV, Azeva TN, Lepesheva GI, Usanov SA (2005) Role of positively charged residues lys267, lys270, and arg411 of cytochrome p450scc (CYP11A1) in interaction with adrenodoxin. Biochemistry (Mosc) 70:664-671.

8. Usanov SA et al. (2002) Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling. Biochemistry 41:8310-8320

9. Strushkevich NV, Harnastai IN, Usanov SA (2010) Mechanism of steroidogenic electron transport: role of conserved Glu429 in destabilization of CYP11A1-adrenodoxin complex. Biochemistry (Mosc) 75:570-578