Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations

John C. Huntera, Anuj Manandhara, Martin A. Carrascoa, Deepak Gurbania, Sudershan Gondia,

Kenneth D. Westovera,1

aDepartments of Biochemistry and Radiation Oncology, The University of Texas Southwestern

Medical Center at Dallas, Dallas, Texas 75390

Supplementary Material

Supplemental Methods:

Protein Expression and purification

An e. coli expression construct encoding codon-optimized wild-type KRAS including a TEV

cleavable N-terminal 6xhis tag in the pJExpress vector was synthesized (DNA2.0). Point

mutations were generated using the GeneArt® site-directed mutagenesis system (Life

Technologies). Protein was expressed and purified as described previously (1). Briefly, BL21

cells were transformed and grown in Luria broth (LB) to an OD600 of ~0.8 at which point protein

expression was induced by the addition of 250µM Isopropyl β-D-1-thiogalactopyranoside

(IPTG). Cells were grown overnight at 16 ºC, pelleted, then resuspended into lysis buffer, 20

mM sodium phosphate (pH 8.0), 500 mM NaCl, 10 mM imidazole, 1 mM 2-mercaptoethanol

(BME), 5% (vol/vol) glycerol with 1mg/mL lysozyme. Protein was purified over an IMAC

cartridge (BioRad) following standard protocols, eluted with 250 mM immidizole, and exchanged

into crystallization buffer ([20 mM Hepes (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT)

over a desalting column (BioRad). The N-terminal His-tag was removed by TEV protease

digestion followed by reverse IMAC purification. Protein was concentrated over a 10kD cutoff

filter (Amicon) then flash frozen and stored at -80C until use.

An expression construct encoding the catalytic domain (residues 714-1047) of RASA1

(P120GAP) was a gift from Xuewu Zhang, PhD. The cDNA sequence of RAS-binding domain of

RAF-1 kinase (residues 51 to 191) with a C-terminal TEV cleavable 6Xhis tag was synthesized

(Mr. Gene) and cloned into a pTriEx expression vector. Expression and purification of P120GAP

and RAF-RBD was performed as described above for KRAS. Protein was buffer exchanged

into 20 mM Tris pH 8.0, 50 mM NaCl, 1 mM DTT, and stored at -80ºC until use.

Nucleotide exchange assay

Proteins were exchanged with 5 mM GDP in 20 mM Tris, 150 mM NaCl at a concentration of 45

µM (1 mg/mL) at 25°C for 60 minutes with gentle shaking. Upon completion of exchange,

protein samples were desalted using 7,000 Da MWCO 0.5 mL Zeba Spin Desalt Columns

(Thermo Scientific, Rockford, IL) according to manufacturer’s guidelines. Proteins were then

concentrated using Amicon Ultra 0.5 mL filters (10,000 Da MWCO; EMD Millipore, Darmstadt,

Germany). Protein concentration was assessed using a Bradford assay and BSA for the

standard curve. If needed, protein concentrations were adjusted to 45 µM (1 mg/mL) with 20

mM Tris, 150 mM NaCl. To validate complete GDP loading, 100 µL of each sample was mixed

with an equal part of 12 M urea, and run on an HPLC column (NP-10) using a sodium acetate

gradient. Specifically, 100 µL of each sample was injected into the column on an Agilent

Technologies 1260 Infinity HPLC instrument (Santa Clara, CA). A step gradient with 1 M NaOAc

was used over 25 minutes and the traces were compared to those of known standards. The

area under the curve (AUC) was calculated and was used to determine percent loading. For the

kinetic nucleotide association assay, 1.5 µM mant-GTP or mant-GDP (in 20 mM Tris, 50 mM

NaCl, 10 mM MgCl2, 20 mM EDTA) and KRAS protein at a final concentration of 750 nM was

added to a 4 mL cuvette. Fluorescence was measured every 1 sec. for 15 minutes at

excitation/emission set to 360 nm/440 nm in a Synergy Neo reader (BioTek). Data was exported

and analyzed using Graphpad Prism (GraphPad Software, Inc., La Jolla, CA). All readings were

performed in triplicate.

GTPase assay

GTPase activity was measured using EnzCheck phosphate assay system (Life Technologies) to

continuously measure phosphate release following the manufacturer’s recommended protocol.

Briefly, KRAS proteins in assay buffer (30 mM Tris, pH 7.5, 1 mM DTT) were loaded with GTP

by incubating with 2.5 mM GTP (25-fold excess) for 2 hours at 20ºC in the presence of 10 mM

EDTA. Protein was desalted over a zeba spin column (Thermo) to remove excess GTP and

protein concentration adjusted to 2 mg/mL. The assay was performed in a clear 384-well plate

(Costar) by mixing 50 µL protein (50 µM final concentration), 20 µL MESG (200 µM), and 5 µL

purine nucleotide phosphorylase (0.5U). GTP hydrolysis was initiated by the addition of 25 µL

assay buffer with 40 mM MgCl2 in the case of intrinsic or 25 µL P120GAP in assay buffer with

40 mM MgCl2 for GAP-stimulated assays. The absorbance at 360nm was read on an eon plate

reader (BioTek) every 8-15 seconds for 1000 seconds at 20ºC. The phosphate concentration at

each point was determined by comparison with a phosphate standard curve and plotted against

time. The hydrolysis rate constant was determined by fitting the data to a single-phase,

exponential non-linear regression curve with the equation [Pi]=[Pi]0 + ([Pi]final-[Pi]0)(1-exp(-kt)) in

Prism 6.05 (Graphpad).

RAF kinase interaction assay

KRAS:RAF kinase interaction assays were performed as previously described (2). Briefly, an N-

terminal flag-tagged KRAS construct was prepared using site-directed mutagenesis and the

protein expressed and purified as described. Purified RAF kinase RBD was labeled with

maleimide PEG biotin (Pierce) following the manufacture’s recommended protocol and labeling

was verified by HABA-dye based avidin binding assay. Purified flag-tagged KRAS (1mg/mL)

and KRAS mutants were loaded with GMPPNP (Sigma-Aldrich) by incubating for 2 hours at

25ºC with a 50-fold excess of nucleotide in the presence of alkaline phosphatase (Thermo-

Fisher). RAF-RBD-biotin was diluted to a final concentration of 40nM and Flag-KRAS to 10nM in

assay buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MgCl2, 5% glycerol, 0.5% BSA) and

added to individual wells of a low-volume white 384-well plate (Perkin Elmer). A 1:3 dilution

series (2000 nM to 0.5 nM) of each mutant KRAS protein was prepared in assay buffer and

added to the corresponding well. The assay was developed by addition of streptavidin donor

and anti-flag acceptor AlphaScreen beads (10 µg/mL) followed by an overnight incubation at

4ºC. Alpha signal was measured in a Neo plate reader (BioTek) with the default AlphaScreen

settings. Data was plotted and fit to a four-parameter non-linear regression line in Prism 6.05

(GraphPad Software, San Diego California USA, www.graphpad.com) to determine relative

affinities.

KRAS x-ray crystal structure determination

Crystals of KRAS mutants grew from hanging vapor diffusion drops with various solutions in the

reservoir: 0.2 M sodium acetate pH 4.5, 0.1 M Tris pH 8.5, 28% PEG 3,350 (G12R), 0.2 M

sodium acetate pH4.5, 0.1 M Tris pH 8.5, 24% PEG 3,350 (G12V), 0.2 M sodium acetate pH

4.5, 0.1 M Tris pH 8.5, 26% PEG 3,350 (G13D, Q61L), 0.1 M MMT pH 4.0, 24% PEG 6000.

Crystals were cryoprotected in 15% glycerol and flash frozen in liquid nitrogen. Diffraction

images were collected at the advanced photon source beamline 19-ID. Data was integrated and

scaled using HKL2000/3000 packages (3, 4). Molecular replacement was performed with 4OBE

as the search model using Phaser software. Manual and automated model building and

refinement were performed using Phenix package and coot software (5, 6). Figure images were

prepared using Pymol (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger,

LLC). Final model and scaled reflection data was deposited at the protein databank. Final

collection and refinement statistics are presented in table S1.

Modeling of KRAS G12R bound to RAF kinase was done by aligning the G12R structure onto

the structure of HRAS bound to the Ras binding domain of RAF kinase (PDB ID 4G0N) using

Pymol software. The coordinates of G12 were replaced with the coordinates of R12 to generate

a .pdb model file. Images were prepared within Pymol. This same procedure was followed to

generate a model of G12R RAS in the hydrolysis transition state from the structure of HRAS

bound to P120GAP (PDB ID 1WQ1).

Electrostatic maps of WT KRAS (PDB ID 4OBE) and KRAS G13D were generated using the

PDB2PQR server (7, 8) with the PARSE force field and PROPKA to assign protonation states.

Images were prepared using the Pymol APBS tools plugin (9).

CCLE data analysis

We downloaded pharmacological profiles for 24 compounds tested against 504 cell lines from

the CCLE database (10). We analyzed the results of cell lines treated with two different MEK

inhibitors, AZD6244, PD-0325901, and two RAF inhibitors, PLX4720, and RAF265. Using the

sequencing data included for each cell line, we categorized each based on KRAS status,

wildtype or mutant. We removed from this analysis any cell lines which harbored mutations in

BRAF, HRAS, NRAS, EGFR, PI3K, or PTEN. We then calculated the mean reported IC50 for

each group, wildtype vs. mutant KRAS, to determine the effect of KRAS mutations on sensitivity

to MEK inhibition. Of the 4 compounds, only one, PD-0325901, had a significantly lower IC for

Mutant KRAS cell lines compared with wildtype KRAS. For this compound, we further stratified

the cell lines into specific KRAS mutations and plotted mean IC50 values for each.

Supplemental References

1. Hunter JC, Gurbani D, Ficarro SB, Carrasco MA, Lim SM, Choi HG, et al. In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C. Proc Natl Acad Sci U S A. 2014;111:8895-900.

2. Lim SM, Westover KD, Ficarro SB, Harrison RA, Choi HG, Pacold ME, et al. Therapeutic targeting of oncogenic k-ras by a covalent catalytic site inhibitor. Angew Chem Int Ed Engl. 2014;53:199-204.

3. Minor ZOaW. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 1997;276:307-26.

4. Minor ZOaW. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 1997;276:307-26.

5. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica Section D, Biological crystallography. 2010;66:213-21.

6. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta crystallographica Section D, Biological crystallography. 2010;66:486-501.

7. Dolinsky TJ, Czodrowski P, Li H, Nielsen JE, Jensen JH, Klebe G, et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic acids research. 2007;35:W522-5.

8. Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic acids research. 2004;32:W665-7.

9. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A. 2001;98:10037-41.

10. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483:603-7.

Figure S1. –GTP hydrolysis rate is stimulated by P120GAP. Wildtype KRAS was loaded with GTP and mixed with the indicated concentration of P120GAP to stimulate GTP hydrolysis. The rate of hydrolysis was determined by continuously measuring phosphate release using a purine nucleoside phosphorylase based colorimetric assay. The concentration of phosphate released vs. time was plotted and the first-order rate constants determined by for each concentration of P120GAP.

Figure S2. – Model of Intrinsic GTP hydrolysis.

A. Published structure of HRAS bound to P120GAP (PDB ID 1WQ1) suggests a central role for Q61 in coordinating with a nucleophilic water molecule during GTP hydrolysis. This coordination is also likely important in GAP-independent GTP hydrolysis by KRAS based on the decrease in rate of hydrolysis observed in Q61 mutants.

B. Modeling of the G12R KRAS structure onto the wildtype HRAS structure (from A) demonstrates that insertion of arginine at position 12 is predicted to clash with Q61 and may prevent proper orientation of the nucleophilic water molecule, perhaps explaining the decrease in rate of intrinsic GTP hydrolysis observed in this mutant.

Data Collection G12V (4TQ9) G12R (4QL3) G13D (4TQA) Q61L (4WA7)Source APS 19-1D APS 19-1D APS 19-1D APS 19-1D

Wavelength (Å) 0.97924 0.97924 0.97924 0.97924Space Group C2 P 21 21 21 C2 P 63

Unit Cella, b, c (Å) 65.3, 41.4, 115.1 39.1, 40.9, 91.8 66.2, 41.3, 114.5 82.49, 82.49, 40.76α, β, γ (°) 90, 105.9, 90 90, 90, 90 90, 105, 90 90, 90, 120

Resolution (Å) 37 – 1.49 24.5-1.04 27.2-1.13 35-1.99Unique Reflections 48,032 68,913 111,363 11,062

Redundancy 4.5 (3.4) 4.6 (2.8) 4.4 (4.0) 5.6 (4.4)Completeness (%) 98.5 (93.6) 96.6 (81.8) 99.7 (99.3) 98.4 (93.0)

R-merge 0.06 (0.50) 0.07 (0.56) 0.05 (0.52) 0.05 (0.57)<I/σ> 28.3 (2.0) 30.8 (1.9) 34.6 (2.0) 43.1 (1.9)

Wilson B-factor (Å2) 15 7.8 11.7 38RefinementResolution 37-1.49 24-1.04 27-1.13 35-1.99

Reflections Used 46,048 68,913 109,383 10,054Reflections for R-Free 1984 1929 1980 1008Non-Hydrogen Atoms 3122 1638 3188 1295

Protein 2758 1397 2764 1256Water 364 241 424 39R-work 0.166 0.130 0.140 0.185R-free 0.194 0.155 0.169 0.233

RMS deviationsBond lengths (Å) 0.009 0.011 0.011 0.005Bond Angles (°) 1.297 1.452 1.476 0.931

Average B-factor (Å2) 21.0 12.0 18.0 56Ramachandran plot (%)

favored/allowed/disallowed 99.1/0.9/0.0 98.2/1.8/0.0 99.7/0.3/0.0 98.0/2.0/0.0MolProbity Score 0.73 (100%) 0.81 (99%) 0.74 (99%) 0.75 (100%)