Human mitogen-activated protein kinase 11 in complex with nilotinib

Target ID:

MAPK11A

Aliases:

MAPK11p38-2P38Bp38BetaP38BETA2PRKM11SAPK2SAPK2B

Deposition Date:

Friday 20th March 2009

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

3GP0

Authors:

Filippakopoulos, P., Barr, A., Fedorov, O., Keates, T., Soundararajan, M., Elkins, J., Salah, E., Burgess-Brown, N., Ugochukwu, E., Pike, A.C.W., Muniz, J., Roos, A., Chaikuad, A., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

3GP0

tabs

Structure Details

MAPK11 (also known as p38β or SAPK2B) is a member of the MAP kinase (MAPK) family and forms together with p38α (also referred to as SAPK2a), p38γ (SAPK3), and p38δ (SAPK4) the p38 MAPK subfamily. It is widely expressed with highest levels in the brain and heart and skeletal muscle (Stein et al., 1997) (Lee et al., 2000).

p38β is activated through its phosphorylation by the dual specificity MAP kinase kinases (MKKs), preferably by MKK6 on the Thr–Gly–Tyr motif, in the activation loop resulting in a conformational change and dimerization (Cuenda and Rousseau, 2007).

p38β is closely related to the well characterised p38α isoform but although the two proteins are similar in sequence (~75% sequence identity) they seem to have different biological functions (Turjanski et al., 2007). Inhibitors currently used to characterise the biological function of p38 do not distinguish between the two isoforms and the specific function of p38β remains to be elucidated. Knockout mice show no apparent phenotype, have no compromised immune system, normal T-cell development and immune responses to TNF and LPS (Beardmore et al., 2005). Additionally, mouse models demonstrated that p38β activity is not required for acute or chronic inflammatory responses and that p38α is the major isoform regulating immune response (O'Keefe et al., 2007). In a mouse model for seizures activation of both the alpha and beta isoform has been described in astrocytes of the hippocampus suggesting a role of p38β in neuronal death and/or astrogliosis (Che et al., 2001). Increased activity of the p38β has also been found in lesions of psoriatic skin compared with nonlesional psoriatic skin and p38β may be a potential target for the treatment of psoriasis (Johansen et al., 2005).

Here we report the structure of the kinase domain of MAPK11 at 1.9 Å resolution in complex with the cancer drug nilotinib.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:5199628

SGC Construct ID: MAPK11A-c007

GenBank GI number: gi|20128774

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTC
TGGTGTAGATCTGGGTACCGAGAACCTGT
ACTTCCAATCCATGCGCGCCGGCTTCTAC
CGGCAGGAGCTGAACAAGACCGTGTGGGA
GGTGCCGCAGCGGCTGCAGGGGCTGCGCC
CGGTGGGCTCCGGCGCCTACGGCTCCGTC
TGTTCGGCCTACGACGCCCGGCTGCGCCA
GAAGGTGGCGGTGAAGAAGCTGTCGCGCC
CCTTCCAGTCGCTGATCCACGCGCGCAGA
ACGTACCGGGAGCTGCGGCTGCTCAAGCA
CCTGAAGCACGAGAACGTCATCGGGCTTC
TGGACGTCTTCACGCCGGCCACGTCCATC
GAGGACTTCAGCGAAGTGTACTTGGTGAC
CACCCTGATGGGCGCCGACCTGAACAACA
TCGTCAAGTGCCAGGCGCTGAGCGACGAG
CACGTTCAATTCCTGGTTTACCAGCTGCT
GCGCGGGCTGAAGTACATCCACTCGGCCG
GGATCATCCACCGGGACCTGAAGCCCAGC
AACGTGGCTGTGAACGAGGACTGTGAGCT
CAGGATCCTGGATTTCGGGCTGGCGCGCC
AGGCGGACGAGGAGATGACCGGCTATGTG
GCCACGCGCTGGTACCGGGCACCTGAGAT
CATGCTCAACTGGATGCATTACAACCAAA
CAGTGGATATCTGGTCCGTGGGCTGCATC
ATGGCTGAGCTGCTCCAGGGCAAGGCCCT
CTTCCCGGGAAGCGACTACATTGACCAGC
TGAAGCGCATCATGGAAGTGGTGGGCACA
CCCAGCCCTGAGGTTCTGGCAAAAATCTC
CTCAGAACACGCCCGGACATATATCCAGT
CCCTGCCCCCCATGCCCCAGAAGGACCTG
AGCAGCATCTTCCGTGGAGCCAACCCCCT
GGCCATAGACCTCCTTGGAAGGATGCTGG
TGCTGGACAGTGACCAGAGGGTCAGTGCA
GCTGAGGCACTGGCCCACGCCTACTTCAG
CCAGTACCACGACCCCGAGGATGAGCCAG
AGGCCGAGCCATATGATGAGAGCGTTGAG
GCCAAGGAGCGCACGCTGGAGGAGTGGAA
GGAGCTCACTTACCAGGAAGTCCTCAGCT
TCAAGCCCTGACAGTAAAGGTGGATACGG
ATCCGAA

Tags and additions: Cleavable N-terminal His6 tag.

Final protein sequence:
mhhhhhhssgvdlgtenlyfq^sMRAGFY
RQELNKTVWEVPQRLQGLRPVGSGAYGSV
CSAYDARLRQKVAVKKLSRPFQSLIHARR
TYRELRLLKHLKHENVIGLLDVFTPATSI
EDFSEVYLVTTLMGADLNNIVKCQALSDE
HVQFLVYQLLRGLKYIHSAGIIHRDLKPS
NVAVNEDCELRILDFGLARQADEEMTGYV
ATRWYRAPEIMLNWMHYNQTVDIWSVGCI
MAELLQGKALFPGSDYIDQLKRIMEVVGT
PSPEVLAKISSEHARTYIQSLPPMPQKDL
SSIFRGANPLAIDLLGRMLVLDSDQRVSA
AEALAHAYFSQYHDPEDEPEAEPYDESVE
AKERTLEEWKELTYQEVLSFKP

^ TEV cleave site

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

Growth medium, induction protocol: 1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 litre of LB containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD₆₀₀ reached ~0.3 then the temperature was adjusted to 18°C. Expression was induced for overnight using 1 mM IPTG at an OD₆₀₀ of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50 mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol; 0.5 mM TCEP.

Extraction buffer, extraction method: Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. PEI (polyethyleneimine) was added to the lysate to a final concentration of 0.15 % and the lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification.

Column 1: Ni-affinity. Ni-NTA (Qiagen), 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% Glycerol, 0.5 mM TCEP; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% glycerol, 0.5 mM TCEP; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol, 0.5 mM TCEP.

Procedure: The cleared lysate was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100 ml binding buffer and 100 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, and 10 mM DTT was added.

Enzymatic treatment: The N-terminal His tag was cleaved by overnight incubation at 4°C with TEV protease. Cleaved products and TEV protease were removed by binding to Ni-NTA after buffer exchange to 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP using a 10 kDa cut-off concentrator.

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

Buffers: 10 mM HEPES, pH 7.5; 100 mM NaCl, 10 mM DTT

Procedure: The protein was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 10 mM HEPES, pH 7.5; 100 mM NaCl, 10 mM DTT using either an ÄKTA prime or ÄKTA express system.

Mass spectrometry characterization: LC- ESI -MS TOF gave a measured mass of 39704 for this construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 11.8 mg/ml using an Amicon 10 kDa cut-off concentrator.

Crystallization: Diffraction quality crystals were grown at 4°C in 150 nl drops from a 1:1 ratio of protein and reservoir solution (20% PEG 3350; 0.1M citrate pH 5.5).

Data Collection: Crystals were cryo-protected using 25% EtGly; Resolution: 1.9 Å resolution limit; X-ray source: Diffraction data were collected at the Diamond beamline I04.

References

Beardmore, V.A., Hinton, H.J., Eftychi, C., Apostolaki, M., Armaka, M., Darragh, J., McIlrath, J., Carr, J.M., Armit, L.J., Clacher, C., et al. (2005). Generation and characterization of p38beta (MAPK11) gene-targeted mice. Molecular and cellular biology 25, 10454-10464.

Che, Y., Yu, Y.M., Han, P.L., and Lee, J.K. (2001). Delayed induction of p38 MAPKs in reactive astrocytes in the brain of mice after KA-induced seizure. Brain research 94, 157-165.

Cuenda, A., and Rousseau, S. (2007). p38 MAP-kinases pathway regulation, function and role in human diseases. Biochimica et biophysica acta 1773, 1358-1375.

Johansen, C., Kragballe, K., Westergaard, M., Henningsen, J., Kristiansen, K., and Iversen, L. (2005). The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin. The British journal of dermatology 152, 37-42.

Lee, S.H., Park, J., Che, Y., Han, P.L., and Lee, J.K. (2000). Constitutive activity and differential localization of p38alpha and p38beta MAPKs in adult mouse brain. Journal of neuroscience research 60, 623-631.

O'Keefe, S.J., Mudgett, J.S., Cupo, S., Parsons, J.N., Chartrain, N.A., Fitzgerald, C., Chen, S.L., Lowitz, K., Rasa, C., Visco, D., et al. (2007). Chemical genetics define the roles of p38alpha and p38beta in acute and chronic inflammation. The Journal of biological chemistry 282, 34663-34671.

Stein, B., Yang, M.X., Young, D.B., Janknecht, R., Hunter, T., Murray, B.W., and Barbosa, M.S. (1997). p38-2, a novel mitogen-activated protein kinase with distinct properties. The Journal of biological chemistry 272, 19509-19517.

Turjanski, A.G., Vaque, J.P., and Gutkind, J.S. (2007). MAP kinases and the control of nuclear events. Oncogene 26, 3240-3253.