Supporting Online Material for

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1 Supporting Online Material for The Structure of a Human p110α/p85α Complex Elucidates the Effects of Oncogenic PI3Kα Mutations Chuan-Hsiang Huang, Diana Mandelker, Oleg Schmidt-Kittler, Yardena Samuels, Victor E. Velculescu, Kenneth W. Kinzler, Bert Vogelstein, Sandra B. Gabelli, L. Mario Amzel *To whom correspondence should be addressed. mamzel@jhmi.edu (L.M.A.); gabelli@jhmi.edu (S.B.G.); vogelbe@jhmi.edu (B.V.) This PDF file includes Materials and Methods Figs. S1 to S3 References Published 14 December, Science 318, 1744 (2007) DOI: /science

2 Supplementary Online Material METHODS Cloning and expression cdna clones of p110α and p85α were obtained from Genecopoeia (Germantown, MD) and Origene (Rockville, MD) respectively. Sequencing revealed several mutations in these clones, and the errors were corrected by site-directed mutagenesis or by PCR using primers containing the wild-type sequence. The p110α cdna was cloned in pfastbac HT A (Invitrogen, Carlsbad, CA) and contained an N-terminal His-Tag (residues -28 to -1; tag: MSYYHHHHHHDYDIPTTENLYFQGAMGS). The p85α cdna was cloned into pfastbac 1 (Invitrogen, Carlsbad, CA). Baculoviruses from these clones were generated with the Bac-to-Bac Baculovirus expression system (Invitrogen, Carlsbad, CA). Baculovirus expression clones were picked and sequenced to ensure their integrity. Sf9 cells were grown in Sf-900 Serum Free Media (Invitrogen) supplemented with 0.5% Penicillin- Streptomycin and incubated at 27 C with no CO 2. For protein expression, twenty 500 cm 2 tissue culture dishes (Corning) were seeded with 1 x 10 8 Sf9 cells per flask. Twenty-four hours after seeding, each dish was co-infected with p110α and p85α viruses at a 1:2 multiplicity of infection ratio. Cells were harvested 72 hours later, and the cell pellet, obtained through centrifugation at 900 x g, was stored at -80 C prior to protein purification. Biochemical activity of the p110α/nish2 heterodimer. The K M for PI/PS was determined by incubating the heterodimer with 10 µm ATP, 0.25 µci/ml γ-p32 ATP, 2.5 mm MgCl 2, and concentrations of L-a-Phosphatidylinositol and 1,2-Diacyl-sn-glycero-3- phospho-l-serine from 9 to 275 um. Lipids were extracted with an organic solvent and the enzymatic incorporation of radiolabel into Phosphatidylinositol-monophosphate was measured by scintillation HUANG ET AL., SOM, PAGE 1

3 counting. The K M for ATP was determined in the same reaction conditions but with 137 µm L-a- Phosphatidylinositol, 154 µm 1,2-Diacyl-sn-glycero-3-phospho-L-serine and ATP concentrations ranging from 2.5 to 80 um. Protein purification Cells were resuspended in lysis buffer (50 mm sodium phosphate ph 8.0, 400 mm NaCl, 5% glycerol, 1% Triton X-100, 10 mm 2-mercaptoethanol, 1 mm orthovanadate, 10 mm imidazole) and lysed by sonication on ice for 1 minute. The lysate was spun down, filtered and loaded onto HisTrap column pre-equilibrated with buffer A (50 mm sodium phosphate ph 8.0, 400 mm NaCl, 10 mm 2- mercaptoethanol, 10 mm imidazole). Target protein complex was eluted by an imidazole gradient with 0-100% buffer B (50 mm sodium phosphate ph 8.0, 400 mm NaCl, 10 mm 2-mercaptoethanol, 250 mm imidazole). The sample was then passed through a desalting column before loading onto MonoQ column pre-equilibrated with binding buffer (50 mm Tris ph 8.5 and 1 mm DTT), followed by a gradient of 0-100% elution buffer (50 mm Tris ph 8.5, 500 mm NaCl, 1 mm DTT). Fractions containing the target protein complex were collected, concentrated to 5 ml, loaded onto Sephacryl 16/60 S-200 column and eluted with gel filtration buffer (50 mm Tris ph 8.0, 100 mm NaCl, and 1 mm DTT). The sample was transfered to a final buffer containing 20 mm Tris ph 8.0, 50 mm NaCl, 1 mm DTT using a desalting column and concentrated to mg/ml. Samples were stored at -80 C. Crystallization Crystallization screening was performed by hanging drops of 400 nl (200 nl protein nl reservoir) at 20 C. After screening about 2000 conditions, needle crystals were first identified in a solution containing 2.0 M sodium formate. Hair seeding was then used to obtain larger crystals in optimized mother liquor (1.4 M sodium formate, 100 mm HEPES ph 7.0). Larger crystals were HUANG ET AL., SOM, PAGE 2

4 selected for macroseeding. Diffraction-quality crystals (0.05 mm x 0.1 mm x 0.6 mm in size) were obtained after repeated rounds of macroseeding over a period of one to two months. Crystals were flash-frozen in liquid nitrogen using 4.0 M sodium formate and 10% glycerol as cryoprotectant. Data collection X-ray diffraction data were collected at beamlines X6A and X29 of NSLS at Brookhaven National Laboratory. The crystals diffracted to resolution ranging from 3.0 ~ 3.5 Å. They belong to space group P , with unit cell parameters a = Å, b = Å, c = Å. Data were processed with HKL2000. Data collection statistics are listed in table 1. Structure determination Molecular replacement using PI3K γ as search model (PDB ID: 1E7U), carried out with AmoRe(S1), successfully identified a solution for the corresponding region of p110α (residues ). Subsequent model building was performed with the program O(S2). Residues in the original search model were replaced by those of p110α and a solvent flipped map was then calculated using the program SOLOMON of the CCP4 Suite(S3). In this map, the density corresponding to the coiled-coil region ish2 (residues of p85) was clearly visible. A polyalanine coiled-coil was built into that region and an electron density map calculated with that model revealed secondary structure elements in the ABD region of p110α (residues 1-108). Polyalanine chains were built to fit the new densities. For further adjustments, the ABD-iSH2 structure was used as a guide (S4)( PDBID: 2V1Y). Through iterated rounds of model building and refinement, the R dropped from 40 % to 29 %. However, parts of the p110α ABD, C2 and p85 ish2 regions had high B factors and poor electron densities. These domains are far away from crystal contact sites and appear to be less ordered. TLS refinement HUANG ET AL., SOM, PAGE 3

5 significantly lowered the B factors in those regions. All the refinement was performed with the program REFMAC5 of the CCP4 Suite(S3). Refinement statistics are shown in Table 1. Other calculations The accessible and buried surface areas in the p110α/nish2 heterodimer were calculated with the program ArealMol of the CCP4 Suite, using a radius of 1.4 Å for probe solvent molecules. Structural alignments were performed with the Protein Structure Comparison Service of the European Bioinformatic Institute(S5) and with LSQman of the Uppsala Software Factory(S6). S1. J. Navaza, Acta Crystallogr A A50, 157 (1994). S2. T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallogr A 47, 110 (1991). S3. Collaborative Computational Project Number 4, Acta Crystallogr D Biol Crystallogr 50, 760 (1994). S4. N. Miled et al., Science 317, 239 (2007). S5. E. Krissinel, K. Henrick, Acta Crystallogr D Biol Crystallogr 60, 2256 (2004). S6. A. T. Jones, G. J. Kleywegt, Proteins Suppl 3, 30 (1999). HUANG ET AL., SOM, PAGE 4

6 β1a β2a α1a M P P R P S S G E L W G I H L M P P R I L V E C L L P N G M I V T L E C L R E A T L I T I K H E L F K E A R p110gss 1 M M E L E N Y E Q P V V L R E D N R R R R R R M K P R S T A A S L S S M E L I P I E F V L P T S Q R N T K T P E T A L L H V A G H G N V E Q M K A Q V W L R A L E T S V S A D F Y H R 1 M M E L E N Y K Q P V V L R E D N C R R R R R M K P R S A A S L S S M E L I P I E F V L P T S Q R K C K S P E T A L L H V A G H G N V E Q M K A Q V W L R A L E T S V A A D F Y H R L β3a β4a β5a α1l α2l 55 K Y P L H Q L L Q D E S S Y I F V S V T Q E A E R E E F F D E T R R L C D L R L F Q P F L K V I E P V G N R E E K I L N R E I G F A I G M P V C E F D M V K D P E V Q D F R R N I L N p110gss 92 L G P D H F L L L Y Q K K G Q W Y E I Y D K Y Q V V Q T L D C L R Y W K V L H R S P G Q I H V V Q R H A S E E T L A F Q R Q L N A L I G Y D V T D V S N V H D D E L E F T R R R L V T 92 G P H H F L L L Y Q K K G Q W Y E I Y D K Y Q V V Q T L D C L R Y W K A T H R S P G Q I H L V Q R H P P S E E S Q A F Q R Q L T A L I G Y D V T D V S N V H D D E L E F T R R G L V T α2l α3l α4l β1r β2r α1r. 146 V C K E A V D L R D L N S P H S R A M Y V Y P P N V E S S P E L P K H I Y N K L D K G Q. I I V V I W V I V S P N N D K Q K Y T L K I N H D C V P E Q V I A E A I R K K T R S M L L S p110gss 183 P R M A E V A G R D... P K L Y A M H P W.... V T S K P L P E Y L L K K I T. N N C V F I V I H R S S Q T I K V S A D D T P G T I L Q S F F T K M A K N E P R M A E V A S R D... P K L Y A M H P W.... V T S K P L P E Y L W K K I A. N N C I F I V I H R S S Q T I K V S P D D T P G A I L Q S F F T K M A K. K K S L 236 S E Q L K L C V L E Y Q G K Y I L K V C G C D E Y F L E K Y P L S Q Y K Y I R S C I M L G R M P N L M L M A K E S L Y S Q L P M D C F T M P S Y S R R I S T A T P Y M N G p110gss R D F V L R V C G R D E Y L V G E T P I K N F Q W V R H C L K N G E E I H L V L D T P P D P A L D E V R K E E W P L V D D C T G V T G Y H E Q L T I H G K D H 259 M D I P E S Q S E Q... D F V L R V C G R D E Y L V G E T P I K N F Q W V R H C L K N G E E I H V V L D T P P D P A L D E V R K E X W P L V D D C T G V T G Y H E Q L T I H G K D H β1c2 β2c2 β3c2 β4c2 β5c2 321 E T S T K. S L W V I N S A L R I K I L C A T Y V N V N I R D I D K I Y V R T G I Y H G G E P L C D N V N T Q R V P C S. N P R W N E W L N Y D I Y I P D L P R A A R L C L S I C S V p110gss 336 E S V F T V S L W D C D R K F R V K I R G I D I P V L P R T A D L T V F V E A N I Q Y G Q Q V L C Q R R T S P K. P F T E E V L W N V W L E F S I K I K D L P K G A L L N L Q I Y C G 347 E S V F T V S L W E C D R K F R V K I R G I D I P V L P R N T D L T V F V E A N I Q H G Q Q V L C Q R R T S P K. P F T E E V L W N V W L E F S I K I K D L P K G A L L N L Q I Y C G. β6c2 β7c2 β8c2 410 K G. R K G A K E E H C P L A W G N I N L F D Y T D T L V S G K M A L N L W P V P H G L E D L L N P I G V T G.. S N P N.. K E T.. P C L E L E F D W FS S p110gss 426 K A P A L S G K T S A E M P S P E S K G K A Q L L Y Y V N L L L I D H R F L L R H G E Y V L H M W Q L. S G K G E D Q G S F N A D K L T S A T N P D K E N S M S. I S I L L D N YC H 437 K A P A L S S K A S A E S P S S E S K G K V Q L L Y Y V N L L L I D H R F L L R R G E Y V L H M W Q I. S G K G E D Q G S F N A D K L T S A T N P D K E N S M S. I S I L L D N YC H T. α6l B1H A2H 483 V V K F P D M S V I E E H A N W S V S R E A G F S Y S H A G L S N R L A R D N E L R E N D K E Q L K A I S T R D P L S E I T E Q E K D F L W S H R H Y C V T I P E I L P K L L L S V K p110gss 515 P I A L P K H R P T P D P E G D R V R A E.. M P N Q L R K Q L E A I I A T D P L N P L T A E D K E L L W H F R Y E S L K D P K A Y P K L F S S V K 526 P I A L P K H.. Q P T P D P E G D R V R A E M P N Q L R K Q L E A I I A T D P L N P L T A E D K E L L W H F R Y E S L K H P K A Y P K L F S S V K B2H A3H B3H A4H A4H B4H 574 W N S R D E V A Q M Y C L V K D W P P..... I K P E Q A M E L L D C N Y P D P M V R G F A V R C L E K Y L T D D K L S Q Y L I Q L V Q V L K Y E Q Y L D N L L V R F L L K K A L T p110gss 587 W G Q Q E I V A K T Y Q L L A K R E V W D Q S A L D V G L T M Q L L D C N F S D E N V R A I A V Q K L E. S L E D D D V L H Y L L Q L V Q A V K F E P Y H D S A L A R F L L K R G L R 598 W G Q Q E I V A K T Y Q L L A R R E V W D Q S A L D V G L T M Q L L D C N F S D E N V R A I A V Q K L E. S L E D D D V L H Y L L Q L V Q A V K F E P Y H D S A L A R F L L K R G L R A5H B5H α1k α2k αk N Q R I G H F F F W H L K S E M H. N K T V S Q R F G L L L E S Y C R A C G. M Y L K H L N R Q V E A M E K L I N L T D I L K Q E K K D E.. T Q K V Q M K F L V E Q M R R P D F M. p110gss 677 N K R I G H F L F W F L R S E I A Q S R H Y Q Q R F A V I L E A Y L R G C G T A M L H D F T Q Q V Q V I D M L Q K V T I D I K S L S A E K Y D V S S Q V I S Q L K Q K L E N.. L Q N 688 N K R I G H F L F W F L R S E I A Q S R H Y Q Q R F A V I L E A Y L R G C G T A M L H D F T Q Q V Q V I E M L Q K V T L D I K S L S A E K Y D V S S Q V I S Q L K Q K L E N.. L Q N.. β1k β2k β3k β4k β5k α4k 746 D A L. Q G F L S P L N P A H Q L G N L R L E E C R I M S S A K R P L W L N W E N P D I M S E L L F Q N N E I I F K N G D D L R Q D M L T L Q I I R I M E N I W Q N Q G L D L R M L P p110gss 766 L N L P Q S F R V P Y D P G L K A G A L V I E K C K V M A S K K K P L W L E F K C A D P T A L S N. E T I G I I F K H G D D L R Q D M L I L Q I L R I M E S I W E T E S L D L C L L P 777 S Q L P E S F R V P Y D P G L K A G A L A I E K C K V M A S K K K P L W L E F K C A D P T A L S N. E T I G I I F K H G D D L R Q D M L I L Q I L R I M E S I W E T E S L D L C L L P β6k β7k β8k α5k α6k α7k β9k 836 Y G C L S I G D C V G L I E V V R N S H T I M Q I Q C K G G L K. G A L Q F N S H T L H Q W L K D K N. K G E I Y D A A I D L F T R S C A G Y C V A T F I L G I G D R H N S N I M V K p110gss 856 Y G C I S T G D K I G M I E I V K D A I A K I Q Q S T V G N T G A F K D E V.. L S H W L K E K C P I E E K F Q A A V E R F V Y S C A G Y C V A T F V L G I G D R H N D N I M I S 867 Y G C I S T G D K I G M I E I V K D A I A K I Q Q S T V G N T G A F K D E V.. L N H W L K E K S P T E E K F Q A A V E R F V Y S C A G Y C V A T F V L G I G D R H N D N I M I T.. β10k α8k α9k α10k 925 D D G Q L F H I D F G H F L D H K K K K F G Y K R E R V P F V L T Q D F L I V I S K G A Q E C. T K T R E F E R F Q E M C Y K A Y L A I R Q H A N L F I N L F S M M L G S G M P E L Q p110gss 945 E T G N L F H I D F G H I L G N Y K S F L G I N K E R V P F V L T P D F L F V M. G T.. S G K K T S L H F Q K F Q D V C V K A Y L A L R H H T N L L I I L F S M M L M T G M P Q L T 956 E T G N L F H I D F G H I L G N Y K S F L G I N H E R V P F V L T P D F L F V M. G T.. S G K K T S P H F Q K F Q D I C V K A Y L A L R H H T N L L I I L F S M M L M T G M P Q L T α2r β3r α3r β4r α5l α11k α12k 1015 S F D D I A Y I R K T L A L D K T E Q E A L E Y F M K Q M N D A H H G G W K M D W I F H T I K Q H A L N p110gss 1033 S K E D I E Y I R D A L T V G K S E E D A K K Y F L D Q I E V C R D K G W T V Q F N W F L H L V L G I S K E D I E Y I R D A L T V G K N E E D A K K Y F L D Q I E V C R D K G W T V Q F N W F L H L V L.....

7 fig. S1. Structural alignment of the sequences of human p110α (top), human p110γ (bottom) and wild boar p110γ (middle). Residues form the ABD, residues the RBD, residues C2, residues the helical domain, and residues 696 to 1068 the kinase domain. Mutation hotspots (Arg38, Arg88, Asn345, Glu453, Glu542, Glu545 and Gln546, Thr1025, Met1043, His1047) are shaded orange. The N-terminal domain, not conserved between p110α and p110γ, is shown with green letters. For the rest of the sequence, identical residues in all three proteins are shown on a colored background (reflecting the domain color in Fig. 1 and linker regions in black) with white letters and similar residues (matrix Blosom62) are shown in colored letters with white background. The activation loop is marked with black stars, the catalytic loop with pink stars, and with pink circles the loops connecting β1c2 and β1c2 (CBR1), β3c2 and β4c2 (CBR2), and β5c2 and β6c2 in p110α (CBR3). The α-helices, β-strands are depicted as helices and arrows for the human p110α and p110γ, respectively, on the top and bottom lines. β-turns are indicated as T's. β1r refers to the first β-strand of the RBD, α1l refers to the first α-helix of the linker between the ABD and RBD domains, etc.

8 A B fig. S2. Intermolecular contact in the p110 α /p85 heterodimer crystal. (A) Ribbon diagram of a p110α/nish2 heterodimer and a neighboring molecule. The two identical heterodimers are shown in different shades of the same colors. The Ras binding domain of the neighboring molecule is shown in blue with an orange back-side for contrast. (B) Molecular surface of the PI3K colored as electrostatic charges showing the Ras binding domain of a neighboring molecule (green ribbon with blue back-side) bound in the kinase domain active site.

9 RBD Helical C2 ABD Kinase ish2 fig S3. Stereo diagram of Cα traces of p110α/nish2 and p110γ (PDB: 1E7U). p110α/nish2 is color-coded as in Fig. 1 and p110γ is shown in dark grey.