Human CDK12-Cyclin K complex with inhibitor

Target ID:

XX05CRK7A

Deposition Date:

Friday 14th August 2015

Families:

Protein Kinase

Related Diseases:

Cancer

Structure type:

protein-protein

Download Coordinates:

5ACB

Authors:

S.E.DIXON CLARKE,J.M.ELKINS,A.C.W.PIKE, S.GOUBIN,C.STRAIN-DAMERELL,P.MAHAJAN,C.TALLANT,R.CHALK.J.KOPEC,F.FITZPATRICK, A.MACKENZIE,H.WIGGERS,N.BURGESS-BROWN,E.P.CARPENTER,F.VON DELFT,C.H.ARROWSMITH,A.M.EDWARDS,C.BOUNTRA,A.BULLOCK

Site:

Oxford

5ACB

tabs

Structure Details

CDK12 (also known as CRK7 or CrkRS) belongs to the cyclin-dependent kinase (CDK) family. Recent work indicates that the CDK12-cyclinK complex phosphorylates RNA Pol II on Ser2 in the heptad repeat sequence Y1S2P3T4S5P6S7. Ser2 phosphorylation is known to promote transcriptional elongation, an activity thought previously to be unique to the CDK9-cyclin T complex (P-TEFb). Human RNA Pol II contain 52 copies of the heptad repeat. CDK12 may preferentially phosphorylate C-terminal repeats since its activity is also coupled to 3' end formation of pre-mRNA. CDK12 maintains genome stability by regulating the expression of a number of genes involved in the DNA damage response, including BRCA1, FANCI and ATR. CDK12 and CDK13 have also been suggested to bind cyclin L1/2 for the control of RNA splicing. Dysregulation of CDK12 has been identified in several cancers, including ovarian, breast and gastric cancer. Small molecule inhibitors of CDK12 may therefore be of interest in oncology. Here we determined the structure of the phosphorylated kinase domain of human CDK12 in complex with human cyclin K and a new selective covalent inhibitor THZ531 refined at 2.7 Å resolution. The structure indicates that THZ531 irreversibly targets a cysteine (Cys1039) located in a C-terminal extension of the kinase domain. In cells, THZ531 substantially decreased the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. CDK12 and CDK13 inhibition may therefore hold promise for the treatment of cancers that utilize aberrant transcription or genomic instability to gain a selective growth advantage.

Materials and Methods

No. XX
XX05CRK7A
CRK7 + CCNK: Human cyclin-dependent kinase 12 (CDK12) in complex with cyclin K (CCNK), and the covalent inhibitor THZ531
PDB-code: 5ACB

CDK12

Entry Clone Source: Gregg Morin, University of British Columbia

Entry Clone Accession: N/A

SGC Construct ID: CRK7A-c021

Amplified DNA sequence:
TACTTCCAATCCATGACAGAAAGCGA
CTGGGGGAAACGCTGTGTGGACAAGT
TTGACATTATTGGGATTATTGGAGAA
GGAACCTATGGCCAAGTATATAAAGC
CAAGGACAAAGACACAGGAGAACTAG
TGGCTCTGAAGAAGGTGAGACTAGAC
AATGAGAAAGAGGGCTTCCCAATCAC
AGCCATTCGTGAAATCAAAATCCTTC
GTCAGTTAATCCACCGAAGTGTTGTT
AACATGAAGGAAATTGTCACAGATAA
ACAAGATGCACTGGATTTCAAGAAGG
ACAAAGGTGCCTTTTACCTTGTATTT
GAGTATATGGACCATGACTTAATGGG
ACTGCTAGAATCTGGTTTGGTGCACT
TTTCTGAGGACCATATCAAGTCGTTC
ATGAAACAGCTAATGGAAGGATTGGA
ATACTGTCACAAAAAGAATTTCCTGC
ATCGGGATATTAAGTGTTCTAACATT
TTGCTGAATAACAGTGGGCAAATCAA
ACTAGCAGATTTTGGACTTGCTCGGC
TCTATAACTCTGAAGAGAGTCGCCCT
TACACAAACAAAGTCATTACTTTGTG
GTACCGACCTCCAGAACTACTGCTAG
GAGAGGAACGTTACACACCAGCCATA
GATGTTTGGAGCTGTGGATGTATTCT
TGGGGAACTATTCACAAAGAAGCCTA
TTTTTCAAGCCAATCTGGAACTGGCT
CAGCTAGAACTGATCAGCCGACTTTG
TGGTAGCCCTTGTCCAGCTGTGTGGC
CTGATGTTATCAAACTGCCCTACTTC
AACACCATGAAACCGAAGAAGCAATA
TCGAAGGCGTCTACGAGAAGAATTCT
CTTTCATTCCTTCTGCAGCACTTGAT
TTATTGGACCACATGCTGACACTAGA
TCCTAGTAAGCGGTGCACAGCTGAAC
AGACCCTACAGAGCGACTTCCTTAAA
GATGTCGAACTCAGCAAAATGGCTCC
TCCAGACCTCCCCCACTGGCAGGATT
GACAGTAAAGGTGGATA

Expressed protein sequence:
MGHHHHHHSSGVDLGTENLYFQSMTE
SDWGKRCVDKFDIIGIIGEGTYGQVY
KAKDKDTGELVALKKVRLDNEKEGFP
ITAIREIKILRQLIHRSVVNMKEIVT
DKQDALDFKKDKGAFYLVFEYMDHDL
MGLLESGLVHFSEDHIKSFMKQLMEG
LEYCHKKNFLHRDIKCSNILLNNSGQ
IKLADFGLARLYNSEESRPYTNKVIT
LWYRPPELLLGEERYTPAIDVWSCGC
ILGELFTKKPIFQANLELAQLELISR
LCGSPCPAVWPDVIKLPYFNTMKPKK
QYRRRLREEFSFIPSAALDLLDHMLT
LDPSKRCTAEQTLQSDFLKDVELSKM
APPDLPHWQD

Vector: pFB-LIC-Bse

Tags and additions: MGHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag.

Cyclin K

Entry Clone Source: MGC

Entry Clone Accession: BC015935

SGC Construct ID: CCNKA-c001

Amplified DNA sequence:
TACTTCCAATCCATGTCAGTAACTTC
AGCAAACCTGGACCACACAAAGCCAT
GTTGGTACTGGGATAAGAAAGACTTG
GCTCATACACCCTCACAACTTGAAGG
ACTTGATCCAGCCACCGAGGCCCGGT
ACCGCCGAGAGGGCGCTCGGTTCATC
TTTGATGTGGGCACACGTTTGGGGCT
ACACTATGATACCCTGGCAACTGGAA
TAATTTATTTTCATCGCTTCTATATG
TTTCATTCCTTCAAGCAATTCCCAAG
ATATGTGACAGGAGCCTGTTGCCTCT
TTCTGGCTGGGAAAGTAGAAGAAACA
CCAAAAAAATGTAAAGATATCATCAA
AACAGCTCGTAGTTTATTAAATGATG
TACAATTTGGCCAGTTTGGAGATGAC
CCAAAGGAGGAAGTAATGGTTCTGGA
GAGAATCTTACTGCAGACCATCAAGT
TTGATTTACAGGTAGAACATCCATAC
CAGTTCCTACTAAAATATGCAAAGCA
ACTCAAAGGTGATAAAAACAAAATTC
AAAAGTTGGTTCAAATGGCATGGACA
TTTGTAAATGACAGTCTCTGCACCAC
CTTGTCACTGCAGTGGGAACCAGAGA
TCATAGCAGTAGCAGTGATGTATCTC
GCAGGACGTTTGTGCAAATTTGAAAT
ACAAGAATGGACCTCCAAACCCATGT
ATAGGAGATGGTGGGAGCAGTTTGTT
CAAGATGTCCCGGTCGACGTTTTGGA
AGACATCTGCCACCAAATCCTGGATC
TTTACTCACAAGGAAAACAACAGATG
CCTCATTGACAGTAAAGGTGGATA

Expressed protein sequence:
MGHHHHHHSSGVDLGTENLYFQSMSV
TSANLDHTKPCWYWDKKDLAHTPSQL
EGLDPATEARYRREGARFIFDVGTRL
GLHYDTLATGIIYFHRFYMFHSFKQF
PRYVTGACCLFLAGKVEETPKKCKDI
IKTARSLLNDVQFGQFGDDPKEEVMV
LERILLQTIKFDLQVEHPYQFLLKYA
KQLKGDKNKIQKLVQMAWTFVNDSLC
TTLSLQWEPEIIAVAVMYLAGRLCKF
EIQEWTSKPMYRRWWEQFVQDVPVDV
LEDICHQILDLYSQGKQQMPH

Vector: pFB-LIC-Bse

Tags and additions: MGHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag.

S. cerevisiae CAK

Entry Clone Source: MRC PPU, University of Dundee (plasmid DU5172)

Entry Clone Accession: U60192

SGC Construct ID: CAK1SCA-s002

Construct DNA sequence:
ggatccAAACTGGATAGTATAGACAT
TACACACTGTCAGTTGGTCAAATCTA
CTAGAACTGCTAGGATTTATAGGTCG
GATACATATGCCATTAAATGTCTAGC
ACTAGATTTCGATATCCCGCCACATA
ACGCCAAATTCGAAGTATCGATA TT
AAACAAACTGGGCAACAAATGTAAGC
ACATCTTACCTCTTCTAGAGTC TAA
GGCTACCGATAATAATGACCTATTGT
TGTTGTTTCCCTTTGAAGAGA TGAA
CCTTTATGAGTTCATGCAAATGCACT
ATAAAAGAGATAGAAGAAAA AAAAA
TCCCTATTACGATTTGCTAAATCCCA
GTATCCCAATTGTTGCGGA CCCCCC
CGTTCAGAAATATACTAATCAATTGG
ACGTCAATCGGTATTCTT TGTCCTT
TTTCCGGCAAATGGTTGAAGGGATTG
CATTCTTACATGAGAAC AAGATCAT
TCACCGCGACATCAAACCGCAAAATA
TCATGCTAACAAACAA TACCAGCAC
CGTATCCCCAAAGTTGTACATAATTG
ATTTTGGCATCTCTT ATGACATGGC
AAATAACTCACAAACAAGTGCGGAAC
CCATGGATAGCAAG GTGACGGATAT
AAGCACAGGAATTTACAAGGCCCCAG
AAGTGCTTTTTGG AGTAAAATGCTA
TGATGGTGGCGTGGACGTGTGGTCGT
TGTTGATAATTA TTTCTCAGTGGTT
CCAGAGAGAAACAAGCCGTATGGGGC
ACGTTCCGGCC ATGATTGATGACGG
CAGCGACGACATGAACTCAGATGGAA
GCGATTTCAG ACTGATTTGCTCAAT
ATTCGAAAAGTTGGGCATACCGTCCA
TTCAGAAAT GGGAAGAGGTTGCGCA
ACACGGCTCGGTTGATGCATTTGTTG
GTATGTTT GGTGCAGATGGCGATGG
CAAGTATGTACTGGACCAGGAGAAAG
ATGTACA GATTAGCATTGTTGAGAG
GAATATGCCTCGACTGGACGAGATTG
CGGATG TCAAAGTCAAGCAGAAGTT
CATTAATTGTATCCTGGGGATGGTTT
CATTT TCACCAAACGAAAGATGGAG
CTGTCAAAGAATCTTGCAAGAATTAG
AAAA GCCATAAgaattc

Expressed protein sequence:
MSYYHHHHHHDYDIPTTENLYFQG
AMGSKLDSIDITHCQLVKSTRTAR
IY RSDTYAIKCLALDFDIPPHNA
KFEVSILNKLGNKCKHILPLLESK
ATDNN DLLLLFPFEEMNLYEFMQ
MHYKRDRRKKNPYYDLLNPSIPIV
ADPPVQKY TNQLDVNRYSLSFFR
QMVEGIAFLHENKIIHRDIKPQNI
MLTNNTSTVSP KLYIIDFGISYD
MANNSQTSAEPMDSKVTDISTGIY
KAPEVLFGVKCYDG GVDVWSLLI
IISQWFQRETSRMGHVPAMIDDGS
DDMNSDGSDFRLICSIF EKLGIP
SIQKWEEVAQHGSVDAFVGMFGAD
GDGKYVLDQEKDVQISIVER NMP
RLDEIADVKVKQKFINCILGMVSF
SPNERWSCQRILQELEKP*

Vector: pFASTBAC.HTb

Tags and additions: MSYYHHHHHHDYDIPTTENLYFQ*G cleavable N-terminal hexahistidine tag.

CDK12/Cyclin K Complex with THZ531 bound

Host: SF9 Spodoptera frugiperda Insect cells

Materials & Methods

Co-expression of CDK12, CCNK and CAK: Sf9 cells were grown in Insect-Xpress media (Lonza), to a density of 2x106cells/mL and were co-infected with recombinant CDK12, CCNK and S. cerevisiae CAK (CAKSC1) baculoviruses (P2 virus stocks; 2 mL of CDK12 virus stock, 1 mL of CCNK virus stock and 2mL CAK virus stock, per 1L of cell culture. Cells were shaken at 95 rpm at 27°C in an Innova shaker. After 72 hours post-infection the cultures were harvested by centrifugation for 25min at 900xg at 4°C. Cell pellet from 1L flasks were made up to 50 mL in binding buffer (50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole). Calbiochem protease inhibitor cocktail set III was added to the cell suspension at a 1:1000 dilution. This was transferred to 50 mL falcon tubes, and stored at -20°C.

Extraction method: The frozen cells were thawed and lysed by ultrasonication (Sonic, Vibra Cell) on ice over 10.5 min at 35% amplitude, with the sonicator pulsing ON for 5 sec and OFF for 10 sec. Polyethylenimine (PEI) was added to a final concentration of 0.5% to precipitate DNA and the cell lysate clarified by centrifugation at 21,500 RPM for 1 hour at 4°C. The supernatant was recovered for purification.

Column 1: Ni-Affinity Chromatography. 5 mL of 50 % nickel-sepharose resin slurry (GE Healthcare) was split and applied onto two 1.5 x 10 cm column. The column was washed with ultra-pure water, then pre-equilibrated with binding buffer.

Buffers:
Binding buffer: 500mM NaCl, 50mM HEPES pH 7.5, 5% Glycerol, 5mM Imidazole, 0.5mM TCEP
Wash buffer: 500mM NaCl, 50mM HEPES pH 7.5, 5% Glycerol, 30mM Imidazole, 0.5mM TCEP
Elution buffer I: 500mM NaCl, 50mM HEPES pH 7.5, 5% Glycerol, 50mM Imidazole, 0.5mM TCEP
Elution buffer II: 500mM NaCl, 50mM HEPES pH 7.5, 5% Glycerol, 100mM Imidazole, 0.5mM TCEP
Elution buffer III: 500mM NaCl, 50mM HEPES pH 7.5, 5% Glycerol, 150mM Imidazole, 0.5mM TCEP
Elution buffer IV: 500mM NaCl, 50mM HEPES pH 7.5, 5% Glycerol, 250mM Imidazole, 0.5mM TCEP

Immobilised metal affinity chromatography procedure: Following centrifugation at 21,5000 rpm for 1 hour at 4°C, the supernatant was filtered and applied by gravity flow onto the Ni-sepharose column. The bound protein was then washed with 100 mL binding buffer and subsequently with 60 mL wash buffer. CDK12/CCNK and CAK protein was then eluted by applying a step gradient of imidazole - using 10 mL fractions of elution buffer with increasing concentration of imidazole (50 mM, 100mM, 150mM and 250 mM). Elution fractions were analyzed by SDS PAGE.

Enzymatic treatment: 0.1mg of TEV protease was added to the Ni-eluted protein to remove the tag. Incubation was overnight at 4°C with 10mM DTT and 10mM Arg/Glu.

Column 2: Ion-exchange chromatography - 5mL Hitrap SP column (GE Healthcare) run on ÄKTA-purifier

Buffer:
Buffer A: 50mM MES, pH 6.5; 0.5mM TCEP
Buffer B: 50mM MES, pH 6.5; 0.5mM TCEP; 1M NaCl

Ion-exchange chromatography: CDK12/CCNK protein from Ni-Affinity Chromatography was buffer exchanged into 50mM MES, pH 6.5; 0.5mM TCEP and loaded onto a 5 mL Hitrap SP column column equilibrated in the same buffer. A linear elution gradient was run from 0-500 mM NaCl over 150 mL, followed by 500 mM-1M NaCl over 20 mL. This stage separated any remaining CAK. CDK12/CCNK protein containing fractions were pooled and the buffer adjusted to 300 mM NaCl, 50 mM HEPES pH 7.5, 0.5mM TCEP during concentration in a 10 kD MWCO Amicon Ultra concentrator.

Treatment of CDK12/CCNK protein with THZ531: The CDK12/CCNK complex was then buffer exchanged in a 10 kDa MWCO Amicon Ultra spin concentrator, into a 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP buffer (gel filtration buffer), to raise the pH of the buffer for the subsequent step and to remove any trace of the DTT added before ion-exchange chromatography, as this sulfhydryl-containing reducing agent may interfere with the covalent inhibitor binding to the CDK12 cysteine residue. Incubation of THZ531 with purified mono-phosphorylated CDK12/CCNK complex resulted in near complete covalent binding, as determined by intact mass spectrometry. CDK12/CCNK protein was concentrated to 3 mL volume and 17 µM concentration. THZ531 was then added at 25 µM. The sample was spun at 13,000rpm for 10 minutes to remove any precipitate. The sample was then kept at 4°C overnight. Binding of the compound to CDK12 was monitored by changes to CDK12 intact mass. The intact mass of the protein was confirmed by Electrospray Ionisation/Time-of-Flight Mass Spectrometry (ESI-MS, Agilent Technologies). The purified protein complex had an experimental mass of 37,685 kDa, as expected from the primary sequence of CDK12. Following THZ531 treatment the CDK12 mass shifted to 40,218 kDa consistent with a +559 Da addition corresponding to the covalent binding of THZ531.

Column 3: Size Exclusion Chromatography - S75 HiLoad 26/60 Superdex column (GE Healthcare) run on ÄKTA-Express.

Buffer:
Gel Filtration buffer: 300 mM NaCl, 50 mM HEPES pH 7.5, 0.5mM TCEP

Gel filtration procedure: Prior to applying the protein, the S75 HiLoad 26/60 Superdex column was washed and equilibrated with gel filtration buffer. The concentrated protein was diluted in gel filtration buffer, to around 3 mL and directly applied onto the equilibrated S75 HiLoad 26/60 Superdex column, and run at a flow-rate of 1 mL/min. Fractions (1.8 mL each) containing the protein were pooled together.

Mass spec characterization: The intact mass of the protein was confirmed by Electrospray Ionisation/Time-of-Flight Mass Spectrometry (ESI-MS, Agilent Technologies). The purified protein complex had an experimental mass of 39,659 and 30,403 kDa, as expected from primary sequences of a monophosphorylated CDK12 and CCNK, respectively. Masses were determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% methanol in water with 0.1% formic acid.

Crystallisation of the CDK12/CCNK complex: For crystallisation trials, the CDK12/CycK/THZ531 complex was concentrated to 5.5 mg/mL buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP. Viable diffraction quality crystals were grown at 20°C in 195 nL sitting drops mixing 75 nL protein solution and 20 nL crystal seed stock with 100 nL of a reservoir solution comprising 0.1 M HEPES pH 7.0, 10% PEG8000, 0.2 M magnesium chloride. Crystals were cryo-protected with mother liquor supplemented with an additional 15% ethylene glycol and vitrified in liquid nitrogen after mounting.

Data Collection:2.7 Å resolution
X-ray source: Diffraction data were collected at 100 K on Diamond Light Source beamline I03.

References

Zhang T, Kwiatkowski N, Olson CM, Dixon-Clarke SE, Abraham BJ, Greifenberg AK, Ficarro SB, Elkins JM, Liang Y, Hannett NM, Manz T, Hao M, Bartkowiak B, Greenleaf AL, Marto JA, Geyer M, Bullock AN, Young RA, Gray NS (2016). Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat Chem Biol. 2016 Aug 29. doi: 10.1038/nchembio.2166. [Epub ahead of print]

Dixon-Clarke SE, Elkins JM, Cheng SW, Morin GB, Bullock AN. (2015). Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding. Sci Rep. 5, 17122.

Joshi PM, Sutor SL, Huntoon CJ, Karnitz LM. (2014). Ovarian Cancer-Associated Mutations Disable Catalytic Activity of CDK12, A Kinase That Promotes Homologous Recombination Repair and Resistance to Cisplatin and Poly(ADP-Ribose) Polymerase Inhibitors. J Biol Chem. 2014 Feb 19. [Epub ahead of print] doi: 10.1074/jbc.M114.551143.

Bajrami I, Frankum JR, Konde A, Miller RE, Rehman FL, Brough R, Campbell J, Sims D, Rafiq R, Hooper S, Chen L, Kozarewa I, Assiotis I, Fenwick K, Natrajan R, Lord CJ, Ashworth A. (2014). Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. Cancer Res. 74, 287-97.

Blazek D. (2012). The cyclin K/Cdk12 complex: an emerging new player in the maintenance of genome stability. Cell Cycle. 11, 1049-50.

Rodrigues F, Thuma L, Klämbt C. (2012). The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity. Development. 139, 1765-76.

Cheng SW, Kuzyk MA, Moradian A, Ichu TA, Chang VC, Tien JF, Vollett SE, Griffith M, Marra MA, Morin GB. (2012). Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II. Mol Cell Biol. 32, 4691-704.

Blazek D, Kohoutek J, Bartholomeeusen K, Johansen E, Hulinkova P, Luo Z, Cimermancic P, Ule J, Peterlin BM. (2011). The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. Genes Dev. 25, 2158-72.

Bartkowiak B, Liu P, Phatnani HP, Fuda NJ, Cooper JJ, Price DH, Adelman K, Lis JT, Greenleaf AL. (2010). CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 24, 2303-16.

Chen HH, Wang YC, Fann MJ. (2006). Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation. Mol Cell Biol. 26, 2736-45.

Ko TK, Kelly E, Pines J. (2001). CrkRS: a novel conserved Cdc2-related protein kinase that colocalises with SC35 speckles. J Cell Sci. 114, 2591-603.