Human Aurora B Kinase in complex with INCENP and VX-680

Target ID:

XX07AURKBA

Deposition Date:

Monday 16th January 2012

Families:

Protein Kinase

Structure type:

protein-ligand

Download Coordinates:

4AF3

Authors:

J.M.ELKINS,M.VOLLMAR,J.WANG,S.PICAUD,C.H.ARROWSMITH,A.EDWARDS, C.BOUNTRA,F.VON DELFT,S.KNAPP

Site:

Oxford

4AF3

tabs

Structure Details

Mammals have three aurora kinases: Aurora A, Aurora B and Aurora C. The Aurora kinases are related to the AGC branch of protein kinases (protein kinase A, protein kinase G, protein kinase C)(Vader and Lens 2008). Part of the activation mechanism for most AGC kinases is binding of a C-terminal "hydrophobic motif", FXXF(T/S)F, to the N-terminal lobe of the kinase. This binding often requires phosphorylation of a serine or threonine residue in the hydrophobic motif. However Aurora kinases, instead of binding to such a hydrophobic motif in their C-terminus, bind other proteins to their N-terminal lobe. One such co-factor for Aurora A is the protein TPX2. For Aurora B and C the C-terminal section of the INCENP protein performs a similar function. For part of the cell cycle Aurora B exists as a part of the larger Chromosomal Passenger Complex (CPC) consisting of Aurora B, INCENP, Survivin and Borealin. This complex is required for proper localisation of Aurora B during mitosis (Vader, Medema et al. 2006).

Due to their role in mitotic regulation, the Aurora kinases are seen as targets for inhibition for treatment of cancers (Keen and Taylor 2002), and many Aurora inhibitors have been developed, including some isoform-specific inhibitors (Manfredi, Ecsedy et al. 2007; Mortlock, Foote et al. 2007; Anderson, Lai et al. 2009; Adams, Adams et al. 2010; Shimomura, Hasako et al. 2010).

The structure of Aurora B has previously been determined from Xenopus laevis, in complex with the Aurora B inhibitor Hesperadin (Sessa, Mapelli et al. 2005). Here we present the structure of human Aurora B kinase in complex with the C-terminal section of INCENP and the pan-Aurora inhibitor VX-680 (Harrington, Bebbington et al. 2004).

Materials and Methods

AURKBA (4AF3) Materials & Methods

Entry Clone Source: Mammalian Gene Collection (IMAGE collection Clone ID 2819846)

SGC Construct ID: AURKBA-c004

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ].

AURKBA DNA sequence:
ATGCACCATCATCATCATCATTCTTC
TGGTGTAGATCTGGGTACCGAGAACC
TGTACTTCCAATCCATGCAGAAGGTG
ATGGAGAATAGCAGTGGGACACCCGA
CATCTTAACGCGGCACTTCACAATTG
ATGACTTTGAGATTGGGCGTCCTCTG
GGCAAAGGCAAGTTTGGAAACGTGTA
CTTGGCTCGGGAGAAGAAAAGCCATT
TCATCGTGGCGCTCAAGGTCCTCTTC
AAGTCCCAGATAGAGAAGGAGGGCGT
GGAGCATCAGCTGCGCAGAGAGATCG
AAATCCAGGCCCACCTGCACCATCCC
AACATCCTGCGTCTCTACAACTATTT
TTATGACCGGAGGAGGATCTACTTGA
TTCTAGAGTATGCCCCCCGCGGGGAG
CTCTACAAGGAGCTGCAGAAGAGCTG
CACATTTGACGAGCAGCGAACAGCCA
CGATCATGGAGGAGTTGGCAGATGCT
CTAATGTACTGCCATGGGAAGAAGGT
GATTCACAGAGACATAAAGCCAGAAA
ATCTGCTCTTAGGGCTCAAGGGAGAG
CTGAAGATTGCTGACTTCGGCTGGTC
TGTGCATGCGCCCTCCCTGAGGAGGA
AGACAATGTGTGGCACCCTGGACTAC
CTGCCCCCAGAGATGATTGAGGGGCG
CATGCACAATGAGAAGGTGGATCTGT
GGTGCATTGGAGTGCTTTGCTATGAG
CTGCTGGTGGGGAACCCACCCTTTGA
GAGTGCATCACACAACGAGACCTATC
GCCGCATCGTCAAGGTGGACCTAAAG
TTCCCCGCTTCTGTGCCCACGGGAGC
CCAGGACCTCATCTCCAAACTGCTCA
GGCATAACCCCTCGGAACGGCTGCCC
CTGGCCCAGGTCTCAGCCCACCCTTG
GGTCCGGGCCAACTCTCGGAGGGTGC
TGCCTCCCTCTGCCCTTCAATCTGTC
GCCTGA

AURKBA Final protein sequence (Tag sequence in lowercase):
smQKVMENSSGTPDILTRHFTIDDFE
IGRPLGKGKFGNVYLAREKKSHFIVA
LKVLFKSQIEKEGVEHQLRREIEIQA
HLHHPNILRLYNYFYDRRRIYLILEY
APRGELYKELQKSCTFDEQRTATIME
ELADALMYCHGKKVIHRDIKPENLLL
GLKGELKIADFGWSVHAPSLRRKTMC
GTLDYLPPEMIEGRMHNEKVDLWCIG
VLCYELLVGNPPFESASHNETYRRIV
KVDLKFPASVPTGAQDLISKLLRHNP
SERLPLAQVSAHPWVRANSRRVLPPS
ALQSVA

(Gln55 to Ala344)
The N-terminal residues, sm, derive from the vector following TEV protease digestion to remove the expression tag.

Entry Clone Source: Andrea Musacchio

SGC Construct ID: INCENPA-c002

Vector: pGTvL1-SGC. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ].

INCENPA DNA sequence:
ATGTCCCCTATACTAGGTTATTGGAA
AATTAAGGGCCTTGTGCAACCCACTC
GACTTCTTTTGGAATATCTTGAAGAA
AAATATGAAGAGCATTTGTATGAGCG
CGATGAAGGTGATAAATGGCGAAACA
AAAAGTTTGAATTGGGTTTGGAGTTT
CCCAATCTTCCTTATTATATTGATGG
TGATGTTAAATTAACACAGTCTATGG
CCATCATACGTTATATAGCTGACAAG
CACAACATGTTGGGTGGTTGTCCAAA
AGAGCGTGCAGAGATTTCAATGCTTG
AAGGAGCGGTTTTGGATATTAGATAC
GGTGTTTCGAGAATTGCATATAGTAA
AGACTTTGAAACTCTCAAAGTTGATT
TTCTTAGCAAGCTACCTGAAATGCTG
AAAATGTTCGAAGATCGTTTATGTCA
TAAAACATATTTAAATGGTGATCATG
TAACCCATCCTGACTTCATGTTGTAT
GACGCTCTTGATGTTGTTTTATACAT
GGACCCAATGTGCCTGGATGCGTTCC
CAAAATTAGTTTGTTTTAAAAAACGT
ATTGAAGCTATCCCACAAATTGATAA
GTACTTGAAATCCAGCAAGTATATAG
CATGGCCTTTGCAGGGCTGGCAAGCC
ACGTTTGGTGGTGGCGACCATCCTCC
AAAATCGAGCTCAGAGAACCTGTACT
TCCAATCCATGGAGGCCCATCCCCGG
AAGCCCATCCCCACCTGGGCCCGAGG
CACCCCGCTCAGCCAGGCTATCATTC
ACCAGTACTACCACCCACCGAACCTT
CTGGAGCTCTTTGGAACCATTCTCCC
ACTGGACTTGGAGGATATCTTCAAGA
AGAGCAAGCCCCGCTATCACAAGCGC
ACCAGCTCTGCTGTCTGGAACTCACC
GCCCCTGCAGTGACAGTAAAGGTGGA
TACTCGAGCGGCCGCATCGTGACTGA
CTGA

INCENPA Final protein sequence (Tag sequence in lowercase):
smEAHPRKPIPTWARGTPLSQAIIHQ
YYHPPNLLELFGTILPLDLEDIFKKS
KPRYHKRTSSAVWNSPPLQ

(Glu835 to Gln903)
The N-terminal residues, sm, derive from the vector following TEV protease digestion to remove the expression tag.

AURKBA expression

Host: BL21(DE3)-R3-pRARE2.

Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.

Growth Medium & Induction Protocol: A number of colonies were used to inoculate 70 ml of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol in a 250 ml baffled shaker flask, which was placed in a 37°C shaker overnight. The next day 4x 15 ml of this starter culture was used to inoculate 4x 1L of TB media containing 40 µg/ml kanamycin in 2L baffled shaker flasks. When the OD₆₀₀ was approximately 1.2, the temperature was reduced to 20°C. After a further 25 minutes the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight.

Cell Harvest: Cells were spun at 5500rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C.

INCENPA expression

Host: BL21(DE3)-R3-pRARE2.

Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure.

Growth Medium & Induction Protocol: A number of colonies were used to inoculate 100 ml of LB media containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol in a 250 ml baffled shaker flask, which was placed in a 37°C shaker overnight. The next day 6x 15 ml of this starter culture was used to inoculate 6x 1L of TB media containing 80 µg/ml ampicillin in 2L baffled shaker flasks. When the OD₆₀₀ was approximately 0.5, the temperature was reduced to 20°C. After a further 25 minutes the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight.

Cell Harvest: Cells were spun at 5500rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C.

Extraction buffer, extraction method: The resuspended cell pellets for AURKBA and INCENPA were thawed, combined, and lysed by high-pressure homogenization. PEI (polyethyleneimine) was added to a final concentration of 0.15 %. The cell debris and precipitated DNA were spun down.

Lysis Buffer: 50 mM Tris pH 7.8, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP, Sigma protease inhibitor cocktail.

Column 1: 10 ml of Ni-Sepharose divided in 2x 2.5cm diameter gravity flow columns.

Column 1 Buffers:
Binding buffer: As Lysis Buffer
Wash buffer: As Lysis Buffer except 40 mM Imidazole.
Elution buffer: As Lysis Buffer except 250 mM Imidazole.

Column 1 Procedure: The supernatant was applied by gravity flow onto the Ni column. The Ni column was washed with Wash buffer and the bound protein was eluted by applying a step gradient of Imidazole (5 ml fractions of Elution buffer supplemented with 50mM, 100mM, 150mM and 250mM Imidazole). Collected fractions were pooled and stored at 4°C.

Growth Medium & Induction Protocol: The clarified supernatant was passed through the column. The column was washed with Binding Buffer and Wash Buffer. 50 ml of Elute Buffer was passed through to elute the protein.

Column 2: 10 ml Glutathione Sepharose in a gravity flow column

Column 2 Procedure: The eluted protein from column 1 was passed through column 2. The column was washed with Binding Buffer and then eluted with 50 ml of Binding Buffer containing 10 mM reduced L-glutathione

TEV protease digestion: TEV protease was added. The sample was left at 4°C overnight.

Column 3: S75 16/60 Gel Filtration

Column 3 Buffers:
Gel Filtration buffer: 25 mM Hepes pH 7.5, 200 mM NaCl, 0.5 mM TCEP

Column 3 Procedure: The protein was concentrated to 5 ml volume and injected onto an S75 16/60 GF column (pre-equilibrated in GF Buffer) at 1.0 ml/min. 1.75 ml fractions were collected.

Column 4: Glutathione Sepharose

Column 4 Procedure: Pooled fractions from the gel filtration were passed through 1 ml of glutathione sepharose.

Column 5: Ni-Sepharose

Column 5 Procedure: The flow-through from the glutathione sepharose was loaded onto 0.7 ml of Ni-sepharose. The column was eluted with 5 ml of GF Buffer containing 20, 40, 60, 80, 100, 120 mM imidazole. The desired protein complex appeared in the 40-120 mM fractions.

Concentration: Compound VX-680 was added to the pooled fractions from Column 5. The sample was twice concentrated to 0.25 ml and diluted to 4 ml with GF Buffer before being concentrated to 0.2 ml volume at which the protein concentration was 6 mg/ml (measured by 280 nm absorbance).

Mass spec characterization (before TEV protease digestions):

	Expected	Observed
AURKBA	36116.3	36204.3, 36284.3, 36359.5, 36439.6 (1-4 phosphorylations)
INCENPA	34753.4	34761.0

Masses are +8 from the expected value, for both proteins (miscalibration of mass spectrometer).

After TEV protease digestion the AURKBA protein was monophosphorylated indicating that the additional phosphorylations were on the purification tag.

Crystallization: The complex was crystallised at 20°C in 300 nl drops from a 2:1 ratio of AuroraB:INCENP:VX-680 (6 mg/ml protein) and reservoir solution (10% w/v PEG3350, 0.2M KSCN, 10% Ethylene Glycol, 0.1M BisTrisPropane pH 6.15).

Data Collection:
The crystals were cryo-protected in reservoir solution with 25% (v/v) ethylene glycol and flash-frozen in liquid nitrogen. X-ray diffraction data was collected at 100 K on beam line I04 at DIAMOND.

References

Adams, N. D., J. L. Adams, et al. (2010). "Discovery of GSK1070916, a potent and selective inhibitor of Aurora B/C kinase." J Med Chem 53(10): 3973-4001.
Anderson, K., Z. Lai, et al. (2009). "Biochemical characterization of GSK1070916, a potent and selective inhibitor of Aurora B and Aurora C kinases with an extremely long residence time1." Biochemical Journal 420(2): 259-265.
Harrington, E. A., D. Bebbington, et al. (2004). "VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo." Nat. Med. 10(3): 262-267.
Keen, N. and S. Taylor (2002). "Aurora-Kinase Inhibitors As Anticancer Agents." Nat. Rev. Cancer 4: 927-936. Manfredi, M. G., J. A. Ecsedy, et al. (2007). "Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase." Proc Natl Acad Sci U S A 104(10): 4106-4111.
Mortlock, A. A., K. M. Foote, et al. (2007). "Discovery, Synthesis, and in Vivo Activity of a New Class of Pyrazoloquinazolines as Selective Inhibitors of Aurora B Kinase." J. Med. Chem. 50: 2213-2224.
Sessa, F., M. Mapelli, et al. (2005). "Mechanism of Aurora B Activation by INCENP and Inhibition by Hesperadin." Mol. Cell 18: 379-391.
Shimomura, T., S. Hasako, et al. (2010). "MK-5108, a Highly Selective Aurora-A Kinase Inhibitor, Shows Antitumor Activity Alone and in Combination with Docetaxel." Molecular Cancer Therapeutics 9(1): 157-166.
Vader, G. and S. M. Lens (2008). "The Aurora kinase family in cell division and cancer." Biochim Biophys Acta 1786(1): 60-72.
Vader, G., R. H. Medema, et al. (2006). "The chromosomal passenger complex: guiding Aurora-B through mitosis." J Cell Biol 173(6): 833-837.