1
Supplementary information
Supplementary materials and methods
Materials
1-palmitoyl-2-oleoylsn -glycero-3-phosphocholine (POPC), porcine brain L-αphosphatidylethanolamine (PE), porcine brain PtdIns(4,5)P
2
and cholesterol were from Avanti Polar Lipids, bovine brain 1,2-diacyl-sn-glycero-3-phospho-L-serine
(PS) and chicken egg white albumin were from Sigma-Aldrich. TAMRA-
Ins(1,3,4,5)P4 (part of the PI3-Kinase Fluorescence Polarization Kit) and diC8-
PtdIns(3,4,5)P
3
were from Echelon (Salt Lake City, TX, USA). The pY2-peptide, derived from mouse PDGFR  , has the sequence 735-
ESDGGpY(740)MDMSKDESIDpY(751)VPMLDMKGDIKYADIE-767 (sequence
ID NP_001139740) and was synthesized by Cambridge Peptides (Cambridge, UK).
PIK-108 was a kind gift of Kevan Shokat (UCSF, USA).
Plasmids and baculoviruses
The p110 subunits were cloned into either the pFastbacHT vector (Invitrogen), which encodes an N-terminal His
6
-tag and a TEV protease cleavage site, or into the pOPINE vector (a modified pTriEx-2 vector, Merck, (Possee et al., 2008)), preserving the same
N-terminal tag. The p85 subunits were cloned into either the pFastbac1 vector
(Invitrogen) or the pOPINE vector, and are untagged. Baculoviruses were generated by either the DH10Bac system (Invitrogen) for derivatives of pFastbacHT or by the
Bac10:KO1629 system (Zhao et al., 2003) for derivatives of the pOPINE vectors.
The sources of the constructs are as follows: p110  (mouse, Upstate, NP_032865), p110  (mouse, NP_083370), p110  (mouse, IMAGE clone 4192906, the non-

2 conserved Ser746 insertion was deleted in the final construct), p85  (nic, ic, and i fragments, mouse, IMAGE clone 3979333; ni fragment, human, IMAGE clone
4290954). After TEV cleavage, the p110 proteins have the following extra residues preceding their N-termini: GAMPD (p110  ), GAMGS (p110  ) and GAMDL
(p110  ). Domain boundaries of the p85 fragments are shown in Figure 1. The GST-
(TEV)-Grp1-PH construct was made by subcloning the PH domain (residues 264-
391) from human Grp1 (IMAGE clone 4811560) into the in-house pOPTG vector. All clones were confirmed by sequencing.
Protein expression and purification
All p110/p85 complexes were co-expressed in Sf9 insect cells by co-infection with baculoviruses harbouring the p110 and the p85 subunits (MOI ratio=1:3). Typically, the cells were harvested 48 h post-infection, pelleted and stored frozen. The cells were lysed in a buffer containing 20 mM TrisHCl (pH 8.0), 300 mM NaCl, 30 mM imidazole and 1 mM TCEP by sonication at 4 °C. After ultracentrifugation, the Histagged complex in the clarified lysate was purified via a tandem Ni 2+ (His-Trap) and heparin affinity chromatographic step (GE Healthcare) using a buffer based on 20 mM TrisHCl, pH 8.0 and NaCl. The eluted protein complexes are of high purity as judged by SDS-PAGE. Proteins used for crystallization were further subjected to gel filtration using Superdex 200 columns (GE Healthcare), in a buffer containing 20 mM
TrisHCl (pH 8.0), 100 mM NaK tartrate and 2 mM DTT. Since both N- and Cterminal His
6
-tags affect lipid kinase activity (data not shown), proteins used for functional characterization all had their N-terminal tags removed by TEV protease cleavage, and were either further purified by gel filtration, or, if the yield was too low, exchanged into the final buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM

3 ammonium sulphate and 1 mM TCEP) using centrifugal ultrafiltration units
(Vivaspin, 50 kDa molecular weight cut off membrane). GST-Grp1-PH was expressed in E coli , and purified by glutathione sepharose affinity chromatography followed by gel filtration.
Liposome preparation
Chloroform/methanol solutions of PC, PE, PS, PtdIns(4,5)P
2
and cholesterol were mixed in the desired molar ratio to a final concentration of either 1 mM (for lipid binding assay) or 5 mM (for kinase activity assay), evaporated under a nitrogen gas stream and further dried in a desiccator under vacuum. The lipids were rehydrated in a buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl and 1 mM TCEP (HNT), subjected to bath sonication and 10-20 freeze-thaw cycles between liquid nitrogen and a 40 °C water bath. Small unilamellar vesicles were generated by repeated extrusion through 100 nm pore size polycarbonate filters using a mini-extruder (Avanti Polar
Lipids). The vesicles were measured to have an average diameter of 120 nm by dynamic light scattering (Zetasizer nanoS, Malvern Instruments Inc, USA).
Intrinsic ATPase activity assay
The Transcreener ADP 2 assay kit (Bellbrook Labs, USA) was used to detect ADP production in the absence of lipid substrate. The reactions were carried out at room temperature (25 °C) for 20 min. The reaction mix consisted of 20 mM Hepes (pH
7.5), 150 mM NaCl, 5 mM MgCl
2
, 100  M ATP, 50 nM p110/p85, with or without 2
 M pY2-peptide.
Crystallization and structure determination

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The His
6
-(TEV)-p110  /p85  -niSH2 complex was mixed in 1:1 ratio with the reservoir solution (0.1 M NaK phosphate pH 6.4, 0.14 M (NH
4
)
2
SO
4
, 0.1 M Na formate, 0.42 M Na
2
SO
4
, 15% ethylene glycol). Crystals were cryo-protected in 0.1
M NaKPO4 pH 6.4, 0.14 M (NH
4
)
2
SO
4
, 0.1 M Na formate, 0.4 M Na
2
SO4, and 20% ethylene glycol before flash cooling in liquid nitrogen. The crystals diffracted anisotropically with the best diffraction limit at 3.3 Å. Two data sets, collected on the
ID23-2 beamline at European Synchrotron Radiation Facility (Grenoble, France), and on the I03 beamline at the Diamond Light Source (United Kingdom), were merged to obtain high data multiplicity. The diffraction data were indexed with XDS (Kabsch,
2010) and scaled and merged with Aimless (Phil Evans, unpublished, ftp://ftp.mrclmb.cam.ac.uk/pub/pre/). There is one heterodimer per asymmetric unit in the spacegroup I222. Molecular replacement solution was found using the program
Phaser (McCoy et al., 2007) with the human apo WT p110  /p85  -iSH2 structure
((Huang et al., 2007), PDB ID: 2rd0) as the search model. Rebuilding and refinement were performed with the programs Coot (Emsley and Cowtan, 2004), Refmac5
(Murshudov et al., 2011) and PHENIX (Afonine et al., 2010), using diffraction data to
3.5 Å. Regions that differ from the starting model and required extensive rebuilding included the RBD and C2 domains, the kinase activation loop and C-terminal tail in p110  , and the turn between  1 and  2 of the p85  iSH2. Unlike previously published structures (Huang et al., 2007; Mandelker et al., 2009), the His
6
-(TEV)-tag in our structure does not provide crucial crystal contact and is not observed. Two PIK-
108 molecules were fitted per asymmetric unit, one in the ATP-binding pocket, and one in the kinase C-lobe. The latter was fitted toward the end of model refinement, when the 
A
-weighted F o
-F c electron density maps revealed the shape to be more like
PIK-108 than any other compounds in the crystallization cocktail. More than 96% of

5 the residues in the current model are in the allowed region of the Ramachandran plot.
Crystallographic statistics are presented in Supplementary Table S1.

Supplementary figures
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Figure S1. Comparison of a key element of the kinase domain packing in crystals of the WT p110  /p85  -iSH2 complex with PIK-108 and the H1047R mutant p110  /p85  -niSH2 complex with wortmannin. In both structures, crystal contacts are formed between the kinase C-lobe, including the C-terminal tail, and the RBD from a symmetry-related p110  . The details of crystal contacts differ between the WT and the H1047R mutant structures, with the WT involving the activation loop, and the
H1047R involving residues in the proximity of the ATP-binding site. Nonetheless, the net effect in both structures is to distort part of the C-terminal helix k  11 relative to the WT apo structure, and to stabilize a region corresponding to k  12 that is not observed in the WT apo structure. Neither His1047 (WT) nor Arg1047 (mutant) is directly involved in crystal contacts.

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Figure S2.
Intrinsic ATPase activity in the absence of lipid substrate. Data represent the mean ± s.e. of two independent experiments.

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Figure S3.
Total lipid binding of cancer-linked mutants. Data from flow-cell 1 (Fc1) and Fc1-subtracted data are shown in Figure 3i,j. Protein concentrations=500 nM.

9
Figure S4. Modelling sketchbook for Figure 6. (a) Modelling lipid-binding site in the p110  C2 domain. Crystal structure of the PKC  C2 domain, in complex with two
Ca 2+ ions and a PS molecule with short acyl chains (PDB ID: 1dsy), is superimposed on the p110  C2 domain. This is performed to approximate the phospholipid headgroup position relative to p110  C2, and does not imply that p110  C2 binds PS specifically. ( b ) Modelling ATP binding in the p110  kinase domain. The bound ATP

10 was taken from the p110  /ATP complex after superimposition of p110  kinase domain on that of p110  . ( c ) Modelling helices k  11 and k  12 in the p110  kinase domain. Helices k  11 and k  12 were taken from the p110  /ATP structure after superimposition of k  11 from p110  on that of p110  . ( d ) Modelling of lipid substrate position in p110  kinase domain. Ins(1,4,5)P
3
(IP3), which represents the headgroup of PtdIns(4,5)P
2
, was taken from the structure of inositol 1,4,5trisphosphate 3-kinase complexed with AMPPNP and IP3 (Gonzalez et al., 2004) after superimposition of the bound AMP-PNP on the ATP borrowed from p110  .

Supplementary Table S1 . Statistics of crystallographic data collection and refinement
Data collection
Space Group
Cell dimensions a, b, c (Å)
α, β, γ (°)
Resolution (Å)
R merge
Mean((I)/  (I))
Completeness (%)
Number of measured reflections
Number of unique reflections
Multiplicity
Wilson estimated B factor (Å 2 )
Refinement
R work
R free
(5 % data used)
No. of atoms
Protein
PIK-108 (two molecules)
Mean isotropic B factor (Å 2 )
R.m.s.d. from ideality
Bonds (Å)
Angles (°)
Values in parenthesis are for the highest resolution shell.
I222
136.1, 147.2, 226.5
90, 90, 90
68.1 – 3.5 (3.7-3.5)
0.28 (2.8)
10.2 (1.6)
100 (100)
557699 (90570)
29059 (4621)
19.2 (19.6)
107
0.184
54
103
0.014
1.5
0.228
9508
11

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Supplementary references
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Crystallogr D Biol Crystallogr 60: 2126-2132.
Gonzalez B, Schell MJ, Letcher AJ, Veprintsev DB, Irvine RF, Williams RL. (2004).
Structure of a human inositol 1,4,5-trisphosphate 3-kinase: substrate binding reveals why it is not a phosphoinositide 3-kinase. Mol Cell 15: 689-701.
Huang CH, Mandelker D, Schmidt-Kittler O, Samuels Y, Velculescu VE, Kinzler
KW et al . (2007). The structure of a human p110alpha/p85alpha complex elucidates the effects of oncogenic PI3Kalpha mutations. Science 318: 1744-
1748.
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Mandelker D, Gabelli SB, Schmidt-Kittler O, Zhu J, Cheong I, Huang CH et al .
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Acta Crystallogr D Biol Crystallogr 67: 355-367.
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