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SUPPLEMENTARY INFORMATION doi:10.1038/nature10743 Supplementary Figures and Legends Supplementary Figure 1. CYP17A1 (red boxes) lies at the intersection of steroid hormone biosynthetic pathways. CYP17A1 is involved in both the generation of glucocorticoids and in androgen and estrogen synthesis. CYP17A1 functions as a 17α-hydroxylase converting pregnenolone or progesterone to 17α-hydroxypregnenolone or 17α-hydroxyprogesterone, respectively. Human CYP17A1 can then perform a 17,20-lyase reaction to convert 17α-hydroxypregnenolone to the androgen dehydroepiandrosterone, but does not perform the equivalent reaction on 17α-hydroxyprogesterone. For treatment of androgen- or estrogen-responsive cancers, optimal inhibitors would be 1) selective for CYP17A1 over the many other cytochrome P450 enzymes involved in mineralocorticoid and glucocorticoid synthesis and 2) selective for the 17,20-lyase over the 17α-hydroxylase to reduce undesirable side effects on glucocorticoid synthesis. WWW.NATURE.COM/NATURE 1

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 2. Inhibitors of CYP17A1 used in the present paper. 2 WWW.NATURE.COM/NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 3. The active site of CYP17A1 (purple cartoon and sticks) with TOK-001 (cyan sticks) contains a hydrogen bonding network very similar to that in the structure containing abiraterone. Heme is shown as black sticks with a sphere for iron. Water molecules are shown as small red spheres. The hydrogen bond network is composed of E305, N202, TOK-001, R239, and three water molecules. WWW.NATURE.COM/NATURE 3

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 4. Pregnenolone (blue sticks) docked into CYP17A1 modeled as Fe(IV)=O. The C17 carbon of pregnenolone is 3.6 Å from the oxygen bound to the heme while the C16 carbon is 3.9 Å. The C21 carbon is the closest at 2.9 Å. A hydrogen bond is present from the 3β-OH to residue N202, similar to that observed in the crystal structures with abiraterone and TOK-001. Progesterone docks in essentially the same orientation. 4 WWW.NATURE.COM/NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 5. Structure of CYP17A1 with select functionally relevant mutations shown as sticks. Residues interacting with heme propionates are purple, those on the surface for interaction with cytochrome b 5 are green, and those within the active site are blue. a, Residues shown on the global structure of CYP17A1. Abiraterone is shown as grey sticks. b, Detail of residues (purple) that interact with heme propionates. The only one of these residues that does not directly form a hydrogen bond with one of the heme propionates is D116, which interacts with the backbone of R96. WWW.NATURE.COM/NATURE 5

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 6. Complete synthetic codon-optimized cdna and corresponding amino acid sequence for the truncated, His-tagged CYP17A1Δ19H construct used. Numbers at the end of lines indicate the amino acid number using the full length numbering scheme. ATG GCG AAG AAA ACC GGT GCA AAA TAC CCG AAA TCC TTA CTT AGT CTC CCA M A K K T G A K Y P K S L L S L P 35 CTA GTG GGC TCA CTT CCT TTC TTA CCA CGT CAC GGT CAC ATG CAT AAT AAT L V G S L P F L P R H G H M H N N 52 TTC TTT AAA CTC CAA AAA AAA TAC GGT CCG ATT TAC TCT GTT CGC ATG GGC F F K L Q K K Y G P I Y S V R M G 69 ACC AAA ACC ACC GTC ATT GTC GGC CAT CAT CAG TTA GCT AAG GAA GTA CTC T K T T V I V G H H Q L A K E V L 86 ATC AAA AAA GGC AAA GAT TTC TCC GGA CGC CCA CAA ATG GCT ACC CTC GAT I K K G K D F S G R P Q M A T L D 103 ATT GCG AGC AAC AAC CGT AAA GGC ATC GCT TTC GCT GAC TCA GGC GCT CAT I A S N N R K G I A F A D S G A H 120 TGG CAG TTA CAT CGC CGC TTA GCT ATG GCC ACC TTC GCC TTA TTC AAA GAC W Q L H R R L A M A T F A L F K D 137 GGG GAC CAG AAA CTT GAA AAA ATC ATT TGC CAA GAA ATT TCC ACC TTA TGT G D Q K L E K I I C Q E I S T L C 154 GAC ATG TTA GCA ACG CAT AAC GGC CAG TCT ATT GAT ATT TCC TTT CCA GTA D M L A T H N G Q S I D I S F P V 171 TTT GTT GCA GTT ACC AAT GTT ATT AGT CTC ATC TGT TTC AAC ACA TCT TAC F V A V T N V I S L I C F N T S Y 188 AAA AAT GGC GAT CCT GAA CTG AAC GTG ATT CAG AAC TAT AAC GAA GGT ATT K N G D P E L N V I Q N Y N E G I 205 ATT GAC AAT CTT TCA AAA GAT TCA CTC GTT GAC CTG GTT CCC TGG CTT AAG I D N L S K D S L V D L V P W L K 222 ATC TTC CCA AAC AAA ACC CTT GAA AAA CTT AAA AGT CAT GTA AAA ATC CGT I F P N K T L E K L K S H V K I R 239 AAC GAT TTG CTG AAC AAA ATT CTT GAA AAT TAC AAA GAA AAA TTT CGT TCC N D L L N K I L E N Y K E K F R S 256 GAT TCC ATC ACA AAC ATG TTA GAT ACC CTT ATG CAA GCC AAA ATG AAC TCC D S I T N M L D T L M Q A K M N S 273 GAC AAC GGT AAC GCA GGC CCG GAT CAA GAC TCT GAA CTC CTC TCT GAT AAC D N G N A G P D Q D S E L L S D N 290 CAT ATT CTG ACC ACC ATC GGT GAC ATC TTC GGC GCA GGA GTT GAA ACC ACT H I L T T I G D I F G A G V E T T 307 ACT TCC GTC GTA AAA TGG ACC CTT GCA TTT CTG TTA CAC AAT CCT CAA GTT T S V V K W T L A F L L H N P Q V 324 AAA AAA AAG CTG TAT GAA GAA ATT GAT CAA AAC GTA GGC TTT TCC CGT ACC K K K L Y E E I D Q N V G F S R T 341 6 WWW.NATURE.COM/NATURE

SUPPLEMENTARY INFORMATION RESEARCH CCA ACC ATC TCC GAT CGC AAC CGC CTC TTA CTG CTG GAA GCT ACA ATT CGT P T I S D R N R L L L L E A T I R 358 GAA GTA CTG CGT TTA CGT CCT GTT GCG CCT ATG CTT ATT CCA CAC AAA GCC E V L R L R P V A P M L I P H K A 375 AAC GTT GAT TCA TCA ATT GGG GAA TTC GCC GTG GAC AAA GGT ACC GAA GTC N V D S S I G E F A V D K G T E V 392 ATT ATT AAT CTT TGG GCA CTG CAT CAT AAT GAA AAA GAA TGG CAC CAA CCC I I N L W A L H H N E K E W H Q P 409 GAT CAA TTT ATG CCT GAA CGC TTT CTT AAC CCT GCT GGC ACG CAA CTT ATT D Q F M P E R F L N P A G T Q L I 426 TCC CCA TCT GTT TCT TAT CTT CCT TTT GGC GCT GGA CCA CGT AGC TGT ATT S P S V S Y L P F G A G P R S C I 443 GGT GAA ATC TTG GCA CGT CAG GAA CTG TTT CTT ATT ATG GCA TGG CTC TTG G E I L A R Q E L F L I M A W L L 460 CAA CGC TTT GAC CTG GAA GTC CCA GAT GAT GGC CAA CTT CCC AGC CTG GAA Q R F D L E V P D D G Q L P S L E 477 GGC ATC CCA AAA GTT GTT TTT CTT ATT GAC TCA TTT AAA GTG AAA ATT AAA G I P K V V F L I D S F K V K I K 494 GTT CGT CAG GCC TGG CGC GAG GCT CAG GCG GAA GGT TCA ACC CAT CAC CAC V R Q A W R E A Q A E G S T H H H 511 CAT TAA H * WWW.NATURE.COM/NATURE 7

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Tables Supplementary Table 1. X-ray data collection, refinement, and validation statistics. CYP17A1/Abiraterone CYP17A1/TOK-001 Data collection Space group P2 1 2 1 2 1 P2 1 2 1 2 1 Cell dimensions a, b, c (Å) 85.8, 152.1, 172.9 91.4, 152.1, 167.6 α, β, γ ( ) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å)* 40.49 2.60 (2.67 2.60) 37.08-2.40 (2.46-2.40) Redundancy* 7.5 (7.5) 15.0 (15.1) R pim * 0.060 (0.428) 0.051 (0.463) Mn(I/sd)* 9.6 (2.1) 13.1 (2.9) Completeness* (%) 100.0 (100.0) 100.0 (100.0) Total/Unique Reflections* 524,716 / 70,364 1,379,675 / 92,007 (38556 / 5126) (101,714 / 6,739) Refinement Resolution (Å) 40.49-2.60 37.08-2.40 No. reflections 66,758 87,320 R work / R free 22.2 / 29.1 22.3 / 28.8 Molecules per asymmetric unit 4 4 Residues modeled for each molecule A: 31-273, 283-503 B: 31-274, 283-503 C: 31-502 D: 31-503 A: 30-276, 281-504 B: 31-274, 283-503 C: 31-276, 280-503 D: 30-274, 283-503 Number of atoms / B factor 15,322 / 42.6 15,294 / 46.8 Protein 14,928 / 43.0 14,914 / 47.0 Ligand 104 / 29.6 116 / 35.1 Heme 172 / 27.7 172 / 33.8 Water 118 / 25.4 88 / 40.4 R.m.s deviations Bond lengths (Å) 0.017 0.016 Bond angles (º) 1.768 1.760 Error in coordinates by Luzzati plot (Å) 0.348 0.332 Structure analysis Average RMSD between molecules in the 0.625 0.649 asymmetric unit Average void volume 677.2 651.6 *Highest resolution shell is shown in parenthesis. 8 WWW.NATURE.COM/NATURE

SUPPLEMENTARY INFORMATION RESEARCH Structural Context Ref. Supplementary Table 2. Summary of CYP17A1 mutations, their effects, and structural context. Mutation Identification Effect on CYP17A1 Function * P35L Turkish patient diagnosed 38% of hydroxylation Substitution in N- 44 ΔF53 or ΔF54 Y64S with clinical lyase Japanese patient with combined hydroxylation and lyase Caucasian patient with combined hydroxylation and lyase and 33% lyase Reduced hydroxylase and lyase activities 15% of hydroxylation ; more inactive than active protein No Δ70-77 Italian patients with complete CYP17 K89N Mutagenesis 84% hydroxylase ; 22% lyase G90D Caucasian female No hydroxylase or lyase F93C R96W Patients with combined hydroxylation and lyase Patients with combined hydroxylation and lyase 10% hydroxylase and lyase No hydroxylation or lyase. A105L Mutagenesis study Decrease in 16αhydroxylation S106P Patients with 46,XY disorder of sex no hydroxylase or lyase S106A/T Mutagenesis study 20-30% hydroxylase ; 60% lyase G111S Ile112 duplication F114V Isolated from compound heterozygote patient with complete lack of masculinization; combined hydroxylase/lyase Patient with combined hydroxylation and lyase Patient diagnosed with combined hydroxylase/lyase No hydroxylation or lyase No ; immunoreactive protein produced, but no CO difference spectrum detected Very little hydroxylation or lyase terminus Helix A; may disrupt secondary structure β 1-1 strand, interacts with backbone of F53 in helix A Deletion in β 1-2 strand loop between B and B'; interacts with Ser380 loop between B and B'; larger side chain would disrupt packing loop between B and B'; likely disruption of hydrophobic packing Interacts with heme propionates B'-helix, facing the active site Immediately following B' helix; interacts with L102 backbone Immediately following B' helix; interacts with L102 backbone Loop between B' and C helices; may alter heme packing by altering adjacent D298 that interacts with ligand. Loop between B' and C helices; may alter heme packing and ligand binding Loop between B' and C helices; Interacts directly in active site 45 46 44 47 48 49 50 51 52 53 53 46 54 WWW.NATURE.COM/NATURE 9

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 2. (cont.) Mutation Identification Effect on CYP17A1 Structural Context Ref. Function * D116V R125Q A174E N177D V178D Y201N E305G Patient with congenital adrenal hyperplasia Patient with congenital adrenal hyperplasia German patient with congenital adrenal hyperplasia Turkish patient with congenital adrenal hyperplasia Czech patient with congenital adrenal hyperplasia Infertile patient with mild clinical of 17αhydroxylase/17,20-lyase Patient with isolated lyase Y329D Brazilian patient with 17- hydroxylase ΔE330 or ΔΕ331 I332T P342T R347H R347C ΔL350 or ΔL351 Patient with complete CYP17A1 Same patient as A355T mutation; combination caused ambiguous genitalia Male hermaphrodite patient with combined hydroxylase/lyase Patient with clinical and hormonal findings suggestive of lyase Patient with combined hydroxylase/lyase Korean patient with 17- hydroxylase Little hydroxylation or lyase No hydroxylase or lyase 7% of hydroxylation and very little 17,20-lyase 10% of hydroxylation and lyase No hydroxylase or lyase 31% hydroxylase ; 34% lyase Approximately normal hydroxylase, but very little lyase very low hydroxylase No 15% hydroxylation and 10% lyase 40-45% hydroxylase and lyase activities Retention of hydroxylase but minimal lyase Reduced hydroxylation and no lyase No hydroxylase or lyase Loop between B' and C helices; solvent exposed; interacts with R96 backbone so may indirectly destabilize heme C helix; interacts with heme and P439, S441 backbone E helix; change to Glu may alter hydrophobic packing with L153 E helix; change to Asp would disrupt interaction with E149 E helix; interacts hydrophobically with I145 and I146 in helix D F helix; part of H bonding network interacting with 3βOH I helix; part of H bond network interacting with N202; borders active site J helix; may alter packing and interaction with D464 backbone Middle of J helix, solvent exposed J helix; disruption of hydrophobic packing with surrounding residues Turn between J and J' helices J' helix, solvent exposed; likely cytochrome b 5 binding site J' helix, solvent exposed; likely cytochrome b 5 binding site Turn between J' and K; may disrupt structure 47 55 56 44 56 57 58 59 44 53 60 61 54 62 10 WWW.NATURE.COM/NATURE

SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 2. (cont.) Mutation Identification A355T Isolated from same patient as A332T mutation, in combination caused congenital adrenal hyperplasia R358Q Patient with clinical and hormonal findings suggestive of lyase R362H Mexican patient with 17 hydroxylase with compound heterozygous K110X/R362H mutation R362C Brazilian patient with 17- hydroxylase H373D H373L H373N W406R P409R R416C R416H F417C Patient with congenital adrenal hyperplasia with this mutation and R96W Patients with 17-hydroxyase Japanese patient with combined hydroxylase/lyase Brazilian patient with congenital adrenal hyperplasia Chinese male pseudohermaphrodite with 17- hydroxylase Japanese patient with clinical 17 hydroxylase Patient with congenital adrenal hyperplasia Israeli patient diagnosed with isolated lyase Effect on CYP17A1 Function * Structural Context Ref. No hydroxylase or lyase K helix; may disrupt 53 packing with Ile426 Retention of hydroxylase but minimal lyase No detectable protein in tissues No hydroxylase or lyase No hydroxylation or lyase ; inactive P420 protein by CO difference assay No hydroxylase or lyase No hydroxylase or lyase No hydroxylase or lyase No hydroxylase 8% hydroxylase and 10% lyase No hydroxylase or lyase Originally reported to selectively reduce lyase ; later found to have no or CO difference spectrum K helix; solvent exposed and may interact with cytochrome b 5 Part of conserved ExxR motif in K helix; clear interaction with E359, Q411 backbone; possible interaction with H400, P409 Part of conserved ExxR motif in K helix; clear interaction with E359, Q411 backbone; possible interaction with H400, P409 β 1-4; Interacts directly with heme propionate β 1-4; Interacts directly with heme propionate β 1-4; Interacts directly with heme propionate Loop between K' and K''; alters hydrophobic packing with F417 and Y432 Loop between K' and K''; proline likely important for turn K''; multiple structural H bond interactions K''; multiple structural H bond interactions K''; disruption in hydrophobic packing with W406, Y432 61 63 59 64 65 66 59 67 68 55 69,70,71 WWW.NATURE.COM/NATURE 11

RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 2. (cont.) Mutation Identification Effect on CYP17A1 Function * P428L P434L R440C R440H Brazilian and German patients with 17-hydroxylase Chinese patient combined hydroxylase/lyase Patient with congenital adrenal hyperplasia Patient with hypergonadotropic hypogonadism F453S Patient with very mild 17 hydroxylase symptoms L465P ΔD487, S488, F489 R496H R496C Turkish patient with congenital adrenal hyperplasia Thai patient with 17-hydroxylase Turkish patient with clinical phenotype of isolated 17,20-lyase Patient with clinical phenotype originally reported as isolated 17,20-lyase but actually combined hydroxylase/lyase Residual hydroxylase and no detectable lyase ~6% of 17αhydroxylase No hydroxylation or lyase No hydroxylation or lyase 29% of hydroxylation Very low hydroxylation and lyase No hydroxylase or lyase 30% hydroxylation and lyase Substantially reduced hydroxylation and lyase activities Structural Context Extended loop between K'' and K'''; possible disruption of turn Turn between K'' and K'''; possible disruption of turn K'''; directly interacts with heme and backbone of Ala115 K'''; directly interacts with heme and backbone of Ala115 L helix; disruption of hydrophobic packing with L350 and L353 β 3-3 sheet; disrupts hydrophobic packing with I493 immediately follows β 4-2 ; disruption of fold Single residue between final β 3-2 and C-terminal helix; alter hydrogen bond interactions with D333 Single residue between final β 3-2 and C-terminal helix; alter hydrogen bond interactions with D333 * Unless indicated differently, hydroxylase and lyase refer to the 17α-hydroxylation and 17,20- lyase activities. Table S2 references 44 Biason-Lauber, A. et al. 17α-hydroxylase/17,20-lyase as a model to study enzymatic regulation: Role of phosphorylation. J. Clin. Endocrinol. Metab. 85, 1226-1231 (2000). 45 Yanase, T. et al. Deletion of a phenylalanine in the N-terminal region of human cytochrome P- 450(17α) results in partial combined 17α-hydroxylase/17,20-lyase. J. Biol. Chem. 264, 18076-18082 (1989). 46 Imai, T. et al. Expression and purification of functional human 17α-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17αhydroxylase/17,20-lyase. J. Biol. Chem. 268, 19681-19689 (1993). Ref. 59,72 73 74 75 73 56 76 44 77 12 WWW.NATURE.COM/NATURE

SUPPLEMENTARY INFORMATION RESEARCH 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Auchus, R. J. & Miller, W. L. Molecular modeling of human P450c17 (17α-hydroxylase/17,20- lyase): Insights into reaction mechanisms and effects of mutations. Mol. Endocrinol. 13, 1169 (1999). Yanase, T. 17α-Hydroxylase/17,20-lyase defects. J Steroid Biochem. Mol. Biol. 53, 153-157 (1995). Di Cerbo, A. et al. Combined 17α-hydroxylase/17,20-lyase caused by Phe93Cys mutation in the CYP17 gene. J. Clin. Endocrinol. Metab. 87, 898-905 (2002). Laflamme, N. et al. Mutation R96W in cytochrome P450c17 gene causes combined 17αhydroxylase/17-20-lyase in two French Canadian patients. J. Clin. Endocrinol. Metab. 81, 264-268 (1996). Swart, A. C., Storbeck, K. H., & Swart, P. A single amino acid residue, Ala 105, confers 16αhydroxylase to human cytochrome P450 17α-hydroxylase/17,20 lyase. J. Steroid. Biochem. Mol. Biol. 119, 112-120 (2010). Lin, D., Black, S. M., Nagahama, Y., & Miller, W. L. Steroid 17α-hydroxylase and 17,20-lyase activities of P450c17: Contributions of serine106 and P450 reductase. Endocrinology 132, 2498-2506 (1993). Rosa, S. et al. Clinical, genetic and functional characteristics of three novel CYP17A1 mutations causing combined 17α-hydroxylase/17,20-lyase. Horm. Res. Paediatr. 73, 198-204 (2009). Van Den Akker, E. L. et al. Differential inhibition of 17α-hydroxylase and 17,20-lyase activities by three novel missense CYP17 mutations identified in patients with P450c17. J. Clin. Endocrinol. Metab. 87, 5714-5721 (2002). Ergun-Longmire, B. et al. Two novel mutations found in a patient with 17α-hydroxylase enzyme. J. Clin. Endocrinol. Metab. 91, 4179-4182 (2006). Dhir, V. et al. Steroid 17α-hydroxylase : functional characterization of four mutations (A174E, V178D, R440C, L465P) in the CYP17A1 gene. J. Clin. Endocrinol. Metab. 94, 3058-3064 (2009). Taniyama, M. et al. Subtle 17α-hydroxylase/17,20-lyase with homozygous Y201N mutation in an infertile woman. J. Clin. Endocrinol. Metab. 90, 2508-2511 (2005). Sherbet, D. P. et al. CYP17 mutation E305G causes isolated 17,20-lyase by selectively altering substrate binding. J. Biol. Chem. 278, 48563-48569 (2003). Costa-Santos, M., Kater, C. E., & Auchus, R. J. Two prevalent CYP17 mutations and genotypephenotype correlations in 24 Brazilian patients with 17-hydroxylase. J. Clin. Endocrinol. Metab. 89, 49-60 (2004). Ahlgren, R. et al. Compound heterozygous mutations (Arg 239-stop, Pro 342-Thr) in the CYP17 (P45017α) gene lead to ambiguous external genitalia in a male patient with partial combined 17αhydroxylase/17,20-lyase. J. Clin. Endocrinol. Metab. 74, 667-672 (1992). Geller, D. H., Auchus, R. J., Mendonca, B. B., & Miller, W. L. The genetic and functional basis of isolated 17,20-lyase. Nat. Genet. 17, 201-205 (1997). Hahm, J. R. et al. Functional characterization of mutant CYP17 genes isolated from a 17αhydroxylase/17,20-lyase-deficient patient. Metabolism 53, 1527-1531 (2004). Najera, N. et al. Loss of cytochrome P450 17A1 protein expression in a 17α-hydroxylase/17,20- lyase-deficient 46,XY female caused by two novel mutations in the CYP17A1 gene. Endocr. Pathol. 20, 249-255 (2009). Sahakitrungruang, T., Tee, M. K., Speiser, P. W., & Miller, W. L. Novel P450c17 mutation H373D causing combined 17α-hydroxylase/17,20-lyase. J. Clin. Endocrinol. Metab. 94, 3089-3092 (2009). Monno, S. et al. Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17α-hydroxylase. J. Biol. Chem. 268, 25811-25817 (1993). Katsumata, N., Ogawa, E., Fujiwara, I., & Fujikura, K. Novel CYP17A1 mutation in a Japanese patient with combined 17α-hydroxylase/17,20-lyase. Metabolism 59, 275-278 (2010). WWW.NATURE.COM/NATURE 13

RESEARCH SUPPLEMENTARY INFORMATION 67 68 69 70 71 72 73 74 75 76 77 Lam, C. W. et al., Mutation of proline 409 to arginine in the meander region of cytochrome p450c17 causes severe 17α-hydroxylase. Mol. Genet. Metab. 72, 254-259 (2001). Takeda, Y. et al. Genetic analysis of the cytochrome P-450c17α (CYP17) and aldosterone synthase (CYP11B2) in Japanese patients with 17α-hydroxylase. Clin. Endocrinol. 54, 751-758 (2001). Hershkovitz, E. et al. Homozygous mutation G539R in the gene for P450 oxidoreductase in a family previously diagnosed as having 17,20-lyase. J. Clin. Endocrinol. Metab. 93, 3584-3588 (2008). Gupta, M. K., Geller, D. H., & Auchus, R. J. Pitfalls in characterizing P450c17 mutations associated with isolated 17,20-lyase. J. Clin. Endocrinol. Metab. 86, 4416-4423 (2001). Biason-Lauber, A., Leiberman, E., and Zachmann, M., A single amino acid substitution in the putative redox partner-binding site of P450c17 as cause of isolated 17,20-lyase. J. Clin. Endocrinol. Metab. 82, 3807-3812 (1997). Schwab, K. O. et al. 17α-hydroxylase/17,20-Lyase due to novel compound heterozygote mutations: Treatment for tall stature in a female with male pseudohermaphroditism and spontaneous puberty in her affected sister. J. Pediatr. Endocrinol. Metab. 18, 403-411 (2005). Yang, J. et al. Phenotype-genotype correlation in eight Chinese 17α-hydroxylase/17,20 lyase patients with five novel mutations of CYP17A1 gene. J. Clin. Endocrinol. Metab. 91, 3619-3625 (2006). Patocs, A. et al. Novel mutation of the CYP17 gene in two unrelated patients with combined 17αhydroxylase/17,20-lyase : Demonstration of absent enzyme by expressing the mutant CYP17 gene and by three-dimensional modeling. J. Steroid. Biochem. Mol. Biol. 97, 257-265 (2005). Fardella, C. E., Hum, D. W., Homoki, J., & Miller, W. L. Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17α-hydroxylase. J. Clin. Endocrinol. Metab. 79, 160-164 (1994). Fardella, C. E. et al. Deletion of amino acids Asp487-Ser488-Phe489 in human cytochrome P450c17 causes severe 17α-hydroxylase. J. Clin. Endocrinol. Metab. 77, 489-493 (1993). Yanase, T. et al. Molecular basis of apparent isolated 17,20-lyase : Compound heterozygous mutations in the C-terminal region (Arg(496) Cys, Gln(461) Stop) actually cause combined 17α-hydroxylase/17,20-lyase. Biochim. Biophys. Acta. 1139, 275-279 (1992). 14 WWW.NATURE.COM/NATURE