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

0.97924

Space Group

C2

P 21 21 21

C2

P 63

Unit Cell

a, 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, 40.76

α, 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

35-1.99

Unique 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)

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

38

Refinement

Resolution

37-1.49

24-1.04

27-1.13

35-1.99

Reflections Used

46,048

68,913

109,383

10,054

Reflections for R-Free

1984

1929

1980

1008

Non-Hydrogen Atoms

3122

1638

3188

1295

Protein

2758

1397

2764

1256

Water

364

241

424

39

R-work

0.166

0.130

0.140

0.185

R-free

0.194

0.155

0.169

0.233

RMS deviations

Bond lengths (Å)

0.009

0.011

0.011

0.005

Bond Angles (°)

1.297

1.452

1.476

0.931

Average B-factor (Å2)

21.0

12.0

18.0

56

Ramachandran plot (%)

favored/allowed/disallowed

99.1/0.9/0.0

98.2/1.8/0.0

99.7/0.3/0.0

98.0/2.0/0.0

MolProbity Score

0.73 (100%)

0.81 (99%)

0.74 (99%)

0.75 (100%)