Human Serine/Threonine Kinase 17B (STK17B) in complex with the kinase compound ASC68

Target ID:

STK17BA

Aliases:

DRAK2STK17B

Deposition Date:

Friday 29th January 2010

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

3LM0

Authors:

Ugochukwu, E., Soundararajan, M., Rellos, P., Fedorov, O., Phillips, C., Wang, J., Hapka, E., Filippakopoulos, P., Chaikuad, A., Pike, A.C.W., von Delft, F., Bountra, C., Arrowsmith, C. H., Weigelt, J., Edwards, A., Knapp, S.

Site:

Oxford

3LM0

tabs

Structure Details

STK17B, better known as DRAK2, belongs to the family of death-associated protein kinases. It is ubiquitously expressed with highest expression levels detected in organs of the immune system. DRAK2 is predominantly found in the nucleus but phosphorylation at Ser350 located in the nuclear localization signal induces relocalisation form the nucleus to the cytoplasm (Kuwahara et al., 2008). Little is known so far about DRAK2 substrates and regulation of this kinase. The calcineurin B homologous protein 1CHP1 has been reported to inhibit DRAK2 kinase activity, an effect that is Ca2+-dependent (Nagita et al., 2003). Autophosphorylation at Ser12 is induced by antigen receptor stimulation in T and B cells (Friedrich et al., 2007).

DRAK2 is an inducer of apoptosis and thereby regulates T cell survival, activation and development. Knockout mice for DRAK2 confirm this role and show abnormal T cell numbers and increased T cell proliferation as well as abnormal cytokine physiology (McGargill et al., 2004). These mice have also reduced numbers of germinal centers (GC) due to increase of apoptotic GC B and T cells (Al-Qahtani et al., 2008). Surprisingly, Drak2 knockout mice show reduced susceptibility to some but not all models of automimmune diseases like experimental autoimmune encephalomyelitis (EAE) and automimmune diabetes (Ramos et al., 2008), (McGargill et al., 2008). However, loss of Drak2 does not alter the response to an acute viral infection (McGargill et al., 2008). The resistance to certain autoimmune diseases seems to be due to an increased sensitivity of T cell intrinsic. Accordingly, transgenic mice overexpressing DRAK2 ubiquitously develop augmented apoptosis after TCR stimulation with compromised memory T cell development. Pancreatic islet cells of these animals undergo increased apoptosis upon stimulation with inflammatory cytokines resulting in decreased insulin secretion and glucose intolerance when fed a high-fat diet confirming a role for DRAK2 in the development of diabetes (Gatzka et al., 2009), (Mao et al., 2006). DRAK2 also plays a role in tumorigenesis and the effect of cyclooxygenase-2 (COX-2), which is over-expressed in colorectal cancer to render tumour cells resistant to apoptosis is mediated by negatively regulating DRAK2 (Doherty et al., 2009). Here we report the structure of the kinase domain of STK17B in complex with quercetin at 2.3 Å resolution and with the non-specific inhibitor ASC68 at 2.8 Å respectively.

Figure 1: Co-crystallized ligands.

We would like to thank the group of Prof. Kevan Shokat (University of California San Francisco) for providing the inhibitor ASC68 for co-crystallization studies.

Materials and Methods

Entry Clone Source: FivePrime

Entry Clone Accession: TBA - Opher

SGC Construct ID: STK17BA-c014

GenBank GI number: gi|4758194

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

Coding DNA sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGGAAAACTTTAATAATTTC
TATATACTTACATCTAAAGAGCTAGGGAGA
GGTAAATTTGCTGTGGTTAGACAATGTATA
TCAAAATCTACTGGCCAAGAATATGCTGCA
AAATTTCTAAAAAAGAGAAGAAGAGGACAG
GATTGTCGGGCAGAAATTTTACACGAGATT
GCTGTGCTTGAATTGGCAAAGTCTTGTCCC
CGTGTTATTAATCTTCATGAGGTCTATGAA
AATACAAGTGAAATCATTTTGATATTGGAA
TATGCTGCAGGTGGAGAAATTTTCAGCCTG
TGTTTACCTGAGTTGGCTGAAATGGTTTCT
GAAAATGATGTTATCAGACTCATTAAACAA
ATACTTGAAGGAGTTTATTATCTACATCAG
AATAACATTGTACACCTTGATTTAAAGCCA
CAGAATATATTACTGAGCAGCATATACCCT
CTCGGGGACATTAAAATAGTAGATTTTGGA
ATGTCTCGAAAAATAGGGCATGCGTGTGAA
CTTCGGGAAATCATGGGAACACCAGAATAT
TTAGCTCCAGAAATCCTGAACTATGATCCC
ATTACCACAGCAACAGATATGTGGAATATT
GGTATAATAGCATATATGTTGTTAACTCAC
ACATCACCATTTGTGGGAGAAGATAATCAA
GAAACATACCTCAATATCTCTCAAGTTAAT
GTAGATTATTCGGAAGAAACTTTTTCATCA
GTTTCACAGCTGGCCACAGACTTTATTCAG
AGCCTTTTAGTAAAAAATCCAGAGAAAAGA
CCAACAGCAGAGATATGCCTTTCTCATTCT
TGGCTACAGCAGTGGGACTTTGAAAACTTG
TTTCACCCTGAAGAAACTTCCAGTTCCTCT
CAAACTCAGGATCATTCTGTAAGGTCCTCT
GAAGACAAGACTTCTAAATCCTCCTGACAG
TAAAGGTGGATACGGATCCGAA

Final protein sequence:
MHHHHHHSSGVDLGTENLYFQ^SMENFNNF
YILTSKELGRGKFAVVRQCISKSTGQEYAA
KFLKKRRRGQDCRAEILHEIAVLELAKSCP
RVINLHEVYENTSEIILILEYAAGGEIFSL
CLPELAEMVSENDVIRLIKQILEGVYYLHQ
NNIVHLDLKPQNILLSSIYPLGDIKIVDFG
MSRKIGHACELREIMGTPEYLAPEILNYDP
ITTATDMWNIGIIAYMLLTHTSPFVGEDNQ
ETYLNISQVNVDYSEETFSSVSQLATDFIQ
SLLVKNPEKRPTAEICLSHSWLQQWDFENL
FHPEETSSSSQTQDHSVRSSEDKTSKSS
^ TEV cleave site

Tags and additions: Cleavable N-terminal His6 tag.

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain)

Growth medium, induction protocol: 5 ml from a 50 ml overnight culture containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol were used to inoculate each of two 1 litre cultures of LB containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol. Cultures were grown at 37 °C until the OD₆₀₀ reached ˜0.5 then the temperature was adjusted to 18 °C. Expression was induced overnight using 0.5 mM IPTG at an OD₆₀₀ of 0.9. The cells were collected by centrifugation and the pellet re-suspended in binding buffer and frozen.
Binding buffer: 50 mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5 % glycerol.

Extraction buffer, extraction method: Frozen pellets were thawed and fresh 0.5 mM TCEP added to the lysate. Cells were lysed using C5 high pressure homogenizer (Avestin). The lysate was centrifuged at 16,500 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity. Ni-Sepharose (Amersham), 5 ml of 50 % slurry in 1.5 x 10 cm column, washed with binding buffer.

Buffers:
Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% Glycerol
Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 25 mM Imidazole, 5% glycerol
Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol (step elution).

Procedure: The lysate supernatant was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 30 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted.

Column 2: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad

Buffers: 25 mM HEPES, pH 7.5; 500 mM NaCl, 0.5 mM TCEP

Procedure: The protein was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25 mM HEPES, pH 7.5; 500 mM NaCl, 0.5 mM TCEP using an ÄKTA express system.

Mass spectrometry characterization:LC- ESI -MS TOF revealed that the recombinant protein had a mass of 37286 correlating well with the estimated mass of 37285.3 predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 11 mg/ml using an Amicon 10 kDa cut-off concentrator.

Crystallization: Crystals in complex with ASC68 (K. Shokat, UCSF) were grown at 4 oC in 300 nl sitting drops from a 2:1 ratio of reservoir solution (0.2M (NH₄)₃(cit)20% PEG 3350) and protein.
Crystals in complex with quercetin (CalBioChem, # : 551600) were grown at 4 °C in 300 nl sitting drops from a 2:1 ratio of protein and reservoir solution containing (0.2M NaCl; 0.1M BIS-TRIS pH 5.5; 25% PEG 3350).

Data Collection: Crystals were cryo-protected using the well solution supplemented with 25 % Ethylene glycol and flash frozen in liquid nitrogen.
X-ray source:Diffraction data were collected from a single crystal for each dataset on Diamond beamlines IO4 and IO2 at a single wavelength of 0.976 Å and 0.980 Å respectively.
Phasing: The structures were solved by molecular replacement using the structure of human DAPK (PDB ID 3EHA) as a search model.

References

Al-Qahtani, A., Xu, Z., Zan, H., Walsh, C.M., and Casali, P. (2008). A role for DRAK2 in the germinal center reaction and the antibody response. Autoimmunity 41, 341-352.
Doherty, G.A., Byrne, S.M., Austin, S.C., Scully, G.M., Sadlier, D.M., Neilan, T.G., Kay, E.W., Murray, F.E., and Fitzgerald, D.J. (2009). Regulation of the apoptosis-inducing kinase DRAK2 by cyclooxygenase-2 in colorectal cancer. Br J Cancer 101, 483-491.
Friedrich, M.L., Cui, M., Hernandez, J.B., Weist, B.M., Andersen, H.M., Zhang, X., Huang, L., and Walsh, C.M. (2007). Modulation of DRAK2 autophosphorylation by antigen receptor signaling in primary lymphocytes. J Biol Chem 282, 4573-4584.
Gatzka, M., Newton, R.H., and Walsh, C.M. (2009). Altered thymic selection and increased autoimmunity caused by ectopic expression of DRAK2 during T cell development. J Immunol 183, 285-297.
Kuwahara, H., Nishizaki, M., and Kanazawa, H. (2008). Nuclear localization signal and phosphorylation of Serine350 specify intracellular localization of DRAK2. J Biochem 143, 349-358.
Mao, J., Qiao, X., Luo, H., and Wu, J. (2006). Transgenic drak2 overexpression in mice leads to increased T cell apoptosis and compromised memory T cell development. J Biol Chem 281, 12587-12595.
McGargill, M.A., Choy, C., Wen, B.G., and Hedrick, S.M. (2008). Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease. J Immunol 181, 7593-7605.
McGargill, M.A., Wen, B.G., Walsh, C.M., and Hedrick, S.M. (2004). A deficiency in Drak2 results in a T cell hypersensitivity and an unexpected resistance to autoimmunity. Immunity 21, 781-791.
Nagita, M., Inoue, H., Nakamura, N., and Kanazawa, H. (2003). Two nuclear export signals specify the cytoplasmic localization of calcineurin B homologous protein 1. J Biochem 134, 919-925.
Ramos, S.J., Hernandez, J.B., Gatzka, M., and Walsh, C.M. (2008). Enhanced T cell apoptosis within Drak2-deficient mice promotes resistance to autoimmunity. J Immunol 181, 7606-7616.