Human KRAS (Q61H)

Target ID:

KRASB

Aliases:

C-K-RASK-RAS2AK-RAS2BK-RAS4AK-RAS4BKI-RASKRAS1KRAS2NS3RASK2

Deposition Date:

Monday 23rd April 2007

Families:

GTPases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

3GFT

Authors:

Y.TONG,W.TEMPEL,L.SHEN,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,H.PARK,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

3GFT

tabs

Structure Details

The tumor oncoproteins KRAS, HRAS, NRAS were the first identified and most well-studied members of small GTPases. They share >90% homology in the nucleotide binding domain and also common downstream effectors and upstream GEFs. KRAS, derived from Kirsten RAt Sarcoma viral oncogene homolog, is the most mutated small GTPase involved in numerous cancers and presented many opportunities for therapeutic trials [1]. Two isoforms of KRAS genes, KRAS1 and KRAS2, were initially identified and KRAS1 was later determined to be a pseudogene [2]. Alternative splicing of KRAS2 mRNA results in two variants, isoforms KRASa and KRASb, which differ at the C-terminal region. Posttranslational modification of the two isoforms leads to alternative trafficking pathways and protein localization[3].

Even though the four Ras proteins are highly homologous with each other, gene targeting experiments have shown only KRASb is indispensable for normal mouse embryogenesis [4,5]. Gene know-out experiment indicates a unique role of KRASb in cardiovascular homeostasis[6]. GST pull-down assay has shown calmodulin, a ubiquitous calcium-binding protein, binds only KRASb, but not KRASa, HRAS, or NRAS [7] and modulates its downstream signaling. Cell migration experiment shows that platelet-drived growth factor (PDGF) dependent activation of AKT activation requires KRASb rather than other Ras isoforms[8]. Growth inhibition assay shows RASSF2 is a novel specific effector of KRASb[9]. Understanding the specificity of Ras signaling pathway will be critical in the therapeutic strategy targeting Ras-related human disease including cancers.

We determined the crystal structure of KRAS, isoform b in the active, GppNHp bound form. The overall structure of the KRASb (Fig.1) resembles other known GTPase strucutres. GppNHp is located in the nucleotide binding pocket (Fig.2). A Mg2+ ion stabilize the binding between the protein and GppNHp by coordinating with S17, T35, D57 and the β- and γ- phosphate groups (Fig.3). Quite interestingly, even though residues in the switch II region are identical between KRASb and HRAS, the side chain orientation in this loop, especially that of Y64 is strikingly different in the two proteins.

Materials and Methods

KRASB

PDB:3GFT

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC013572
Entry Clone Source:MGC clone AT9-E8
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with Tev cleavage site: mhhhhhhssgrenlyfqg
Host:E.coli BL21 (DE3) codon plus RIL

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGHEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEK
Vector:pET28a-mhl

Growth

Medium:TB
Antibiotics:
Procedure:The target was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 1.8 L of Terrific Broth medium in the presence of 50 µg/mL kanamycin and chloramphenicol at 37°C. When OD600 was ~3.0, the culture was induced with 1mM IPTG and the temperature was reduced to 15°C, and the cells were allowed to grow overnight before harvesting and flash frozen.

Purification

Procedure
Column 1: HiTrap Ni-NTA column (Pharmacia Amersham)
Column 2: superdex 75.
The lysate was centrifuged at 15000 rpm for 45 min and the supernatant was loaded onto 5 mL HiTrap Ni-NTA column (Pharmacia Amersham) equilibrated with the same binding buffer at 4 ºC using peristaltic pump. The HiTrap Ni-NTA column was steply washed with 25 mL of binding buffer, 25 mL of binding buffer with 30 mM imidazole, and 25 mL of binding buffer with 50 mM imidazole. The His-tagged protein was eluted by linear gradient of imidazole from 50 mM to 500 mM in 50 mL. The eluted protein peak fractions detected by UV280 nm were combined and further purified by gel filtration column superdex 75 with a buffer containing Gel filtration buffer. Protein peak fractions were combined, and DTT was added to a final concentration of 10 mM. Five times molarity of GppNHp is added before concentrating.

Extraction

Procedure
Cultures were centrifuged and the cell pellets were harvested and stored at -80 ºC before use. Cells were thawed and suspended in 150 mL the binding buffer with 0.5% CHAPS (Sigma) and 1 mM phenylmethyl sulfonyl fluoride (PMSF) and lysed with microfluidizer.
Concentration:The protein was concentrated using an Amicon Ultra centrifugal filter and the concentration for protein stock solution was estimated by Bradford to be 45.9 mg/mL.
Ligand
MassSpec:21448.50 Da, expected: 21439.09 Da. The template encodes a mutation Q61H. The measured mass matches with a sequence with H61. This residue is not visible in electron density map.
Crystallization:Crystallization trials were set up using the hanging drop vapor diffusion method at room temperature. The protein drop was equilibrated against a reservoir solution (1:1 volume ratio) containing 20% PEG 3350, 0.2M Lithium Citrate pH 4.0. Crystals reached a size of about 20 microns within one week.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Friday B.B. and Adjei A.A. (2005). K-ras as a target for cancer therapy. Biochim. Biophys. Acta 1756: 127-144.
McGrath J.P., Capon D.J., Smith D.H., Chen E.Y., Seeburg P.H., Goeddel D.V., and Levinson A.D. (1983). Structure and organization of the human Ki-ras proto-oncogene and a related processed pseudogene. Nature 304: 501-506.
Carta C., Pantaleoni F., Bocchinfuso G., Stella L., Vasta I., Sarkozy A., Digilio C., Palleschi A., Pizzuti A., Grammatico P. et al. (2006). Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype. Am. J. Hum. Genet. 79: 129-135.
Plowman S.J., Williamson D.J., O'Sullivan M.J., Doig J., Ritchie A.M., Harrison D.J., Melton D.W., Arends M.J., Hooper M.L., and Patek C.E. (2003). While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable. Mol. Cell Biol. 23: 9245-9250.
Koera K., Nakamura K., Nakao K., Miyoshi J., Toyoshima K., Hatta T., Otani H., Aiba A., and Katsuki M. (1997). K-ras is essential for the development of the mouse embryo. Oncogene 15: 1151-1159.
Potenza N., Vecchione C., Notte A., De R.A., Rosica A., Bauer L., Affuso A., De F.M., RussoT., Poulet R. et al. (2005). Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice. EMBO Rep. 6: 432-437.
Villalonga P., Lopez-Alcala C., Bosch M., Chiloeches A., Rocamora N., Gil J., Marais R., Marshall C.J., Bachs O., and Agell N. (2001). Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling. Mol. Cell Biol. 21: 7345-7354.
Liao J., Planchon S.M., Wolfman J.C., and Wolfman A. (2006). Growth factor-dependent AKT activation and cell migration requires the function of c-K(B)-Ras versus other cellular ras isoforms. J. Biol. Chem. 281: 29730-29738.
Vos M.D., Ellis C.A., Elam C., Ulku A.S., Taylor B.J., and Clark G.J. (2003). RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor. J. Biol. Chem. 278: 28045-28051.