Human cdc-2 like kinase 3 in complex with leucettine L41

Target ID:

CLK3A

Aliases:

FLJ22858PHCLK3PHCLK3/152

Deposition Date:

Monday 28th March 2011

Families:

Protein Kinase

Related Diseases:

SignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

3RAW

Authors:

Filippakopoulos, P., Fedorov, O., King, O., Bullock, A.Muniz, J.R.C., von Delft, F.Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

3RAW

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Structure Details

p>The Clk family consists in human of four kinases that display so-called dual specificity, i.e., they are capable of phosphorylating both Ser/Thr as well as Tyr residues. CLK homologues have been isolated from a number of organisms including Arabidopsis, Drosophila, mouse and human. The kinase domain of CLK family members is located at the C terminus and contains the family signature motif 'EHLAMMERILG', giving rise to the name of these kinases: 'LAMMER' kinases.

It has been shown that CLK family members play a pivotal role controlling splicing by regulating members of the SR family of splicing factors. Both CLK3 and CLK1 have been shown to tightly associate with splicing factors and to phosphorylate them at multiple sites. It is therefore not surprising that inactivating mutation in CLK results in drastic phenotypes. In Drosophila mutation in the Lammer kinase DOA (darkener of apricot) alter sexual differentiation and activity of DOA is required for development of embryonic tissues. In addition, non-functional DOA leads to defects in body segmentation, eye formation, and neuronal development.

High levels of CLK3 expression are found in mature spermatozoa where CLK3 is localized in the spermatozoa acrosome and tail. Interestingly CLK3 is released from sperm during the acrosome reaction suggesting that CLK3 may play role in the fertilization process.

Furthermore it has been shown that expression of all CLK family members is up-regulated during hexamethylene bisacetamide induced murine erythroleukemia cell differentiation and a potential therapeutic use of CLK3 has been suggested for the treatment of frontotemporal dementia and parkinsonism linked to chromosome 17. To provide interesting chemical starting points for CLK3 and models for structure based design we co-crystallized CLK3 with leucettine L41, a potent and selective derivative of the marine sponge 2-aminoimidazolone leucettamine B.

We would like to thanks Prof. L. Meijer (Station Biologique de Roscoff) and Prof. J-P Bazureau (Université de Rennes) for collaboration and for making this compound available.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:5114635

SGC Construct ID: CLK3A-c005

GenBank GI number: gi|4502885

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTC
TTCTGGTGTAGATCTGGGTACCGAGA
ACCTGTACTTCCAATCCATGCAGAGC
AGTAAGCGCAGCAGCCGGAGTGTGGA
AGATGACAAGGAGGGTCACCTGGTGT
GCCGGATCGGCGATTGGCTCCAAGAG
CGATATGAGATTGTGGGGAACCTGGG
TGAAGGCACCTTTGGCAAGGTGGTGG
AGTGCTTGGACCATGCCAGAGGGAAG
TCTCAGGTTGCCCTGAAGATCATCCG
CAACGTGGGCAAGTACCGGGAGGCTG
CCCGGCTAGAAATCAACGTGCTCAAA
AAAATCAAGGAGAAGGACAAAGAAAA
CAAGTTCCTGTGTGTCTTGATGTCTG
ACTGGTTCAACTTCCACGGTCACATG
TGCATCGCCTTTGAGCTCCTGGGCAA
GAACACCTTTGAGTTCCTGAAGGAGA
ATAACTTCCAGCCTTACCCCCTACCA
CATGTCCGGCACATGGCCTACCAGCT
CTGCCACGCCCTTAGATTTCTGCATG
AGAATCAGCTGACCCATACAGACTTG
AAACCAGAGAACATCCTGTTTGTGAA
TTCTGAGTTTGAAACCCTCTACAATG
AGCACAAGAGCTGTGAGGAGAAGTCA
GTGAAGAACACCAGCATCCGAGTGGC
TGACTTTGGCAGTGCCACATTTGACC
ATGAGCACCACACCACCATTGTGGCC
ACCCGTCACTATCGCCCGCCTGAGGT
GATCCTTGAGCTGGGCTGGGCACAGC
CCTGTGACGTCTGGAGCATTGGCTGC
ATTCTCTTTGAGTACTACCGGGGCTT
CACACTCTTCCAGACCCACGAAAACC
GAGAGCACCTGGTGATGATGGAGAAG
ATCCTAGGGCCCATCCCATCACACAT
GATCCACCGTACCAGGAAGCAGAAAT
ATTTCTACAAAGGGGGCCTAGTTTGG
GATGAGAACAGCTCTGACGGCCGGTA
TGTGAAGGAGAACTGCAAACCTCTGA
AGAGTTACATGCTCCAAGACTCCCTG
GAGCACGTGCAGCTGTTTGACCTGAT
GAGGAGGATGTTAGAATTTGACCCTG
CCCAGCGCATCACACTGGCCGAGGCC
CTGCTGCACCCCTTCTTTGCTGGCTT
GACCCCTGAGGAGCGGTCCTTCCACA
CCTAAGACAGTAAAGGTGGATACGGA
TCCGAA

Final protein sequence (Tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^smQS
SKRSSRSVEDDKEGHLVCRIGDWLQE
RYEIVGNLGEGTFGKVVECLDHARGK
SQVALKIIRNVGKYREAARLEINVLK
KIKEKDKENKFLCVLMSDWFNFHGHM
CIAFELLGKNTFEFLKENNFQPYPLP
HVRHMAYQLCHALRFLHENQLTHTDL
KPENILFVNSEFETLYNEHKSCEEKS
VKNTSIRVADFGSATFDHEHHTTIVA
TRHYRPPEVILELGWAQPCDVWSIGC
ILFEYYRGFTLFQTHENREHLVMMEK
ILGPIPSHMIHRTRKQKYFYKGGLVW
DENSSDGRYVKENCKPLKSYMLQDSL
EHVQLFDLMRRMLEFDPAQRITLAEA
LLHPFFAGLTPEERSFHT

^ TEV cleavage site

Tags and additions: Cleavable N-terminal His6 tag.

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

Growth medium, induction protocol: 1ml from a 10 ml overnight culture containing 50µg/ml kanamycin were used to inoculate 1L cultures of LB containing 50µg/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 4 hours using 1 mM IPTG at an OD₆₀₀ of 0.6. The cells were collected by centrifugation, resuspended in 30ml binding buffer, and frozen. The protein was co-expressed with λ-phoshatase.
Lysis buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 mM L-Arg and L-Glu.
Extraction buffer, extraction method: The frozen cells were thawed on ice and binding buffer (plus 1 mM PMSF) were added to a final volume of 50ml. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 18,500prm for 50 minutes and the supernatant collected for purification.

Column 1: Ni-affinity

Column 1 Buffers:
Binding buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 5 mM Imidazole; 50 mM L-Arg and L-Glu.
Wash buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 30 mM Imidazole; 50 mM L-Arg and L-Glu.
Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 50 to 250 mM Imidazole; 50 mM L-Arg and L-Glu.

Column 1 Procedure: In the cold-room, supernatant from the spun-down cell lysate was added to the DE-52 column, the flow through of which was passed onto the nickel resin column. After the lysate had passed through both columns, the columns were washed with approximately 150ml of binding buffer followed by approximately 150ml wash buffer. The flowthrough from the wash was collected and saved. A step-elution was performed by passing buffers with increasing imidazole concentrations over the nickel column and collecting the eluate. 5ml of each buffers containing 50mM, 100mM, 150mM and 250mM imidazole were used. Fractions were collected in 15ml falcon tubes.

Column 2: Size Exclusion Chromatography. Superdex S75 16/60 HiLoad

Column 2 Buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 50 mM L-Arg and L-Glu.

Column 2 Procedure: The protein was concentrated and applied to an S75 16/60 Hi-Load gel filtration column equilibrated in 50 mM HEPES pH 7.5, 500 mM NaCl, 50 mM L-Arg and L-Glu using an ÄKTA Prime system.

Mass spectrometry characterization: LC- ESI -MS TOF gave a measured mass of 44716.2Da for this construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 11.0mg/ml using an Amicon 10kDa cut-off concentrator.

Crystallisation: Crystals grown at 4°C in 150nl sitting drops from a 1:1 ratio of protein to reservoir solution containing 0.1 M BTPropane pH 7.5, 20% PEG3350, 10% EtGly. L41 was co-crystallized with CLK3 by adding the compounds from a 50 mM DMSO stock to the concentrated protein (1 mM end concentration) .

Data collection: Crystals were cryo-protected using the well solution supplemented by 20% ethylene glycol and flash frozen in liquid nitrogen.
X-ray source: Diffraction data were collected from a single crystal at the Swiss Light Source beamline PX10 at a single wavelength of 0.97924Å and the structure was refined to 2.09Å.
Phasing: The structure was solved by molecular replacement using an ensemble of known CLK1/3 structures as a starting model.

References

Gracia-Sacristan, A., Fernandez-Nestosa, M.J., Hernandez, P., Schartzman, J.B., Krimer, DB (2005). Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias towards the skipped splice variant characterizes postcommitment stages. Cell Research 15 :495-503.
Menegay, H., Moeslein, F., Landreth, G. (1999). The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa. Experimental Cell Research 253 :463-473.
Howell BW, Afar DE, Lew J, Douville EM, Icely PL, Gray DA, Bell JC. (1991). STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases. Mol Cell Biol. 11 :568-572.
Myers MP, Murphy MB , Landreth G. (1994). The dual-specificity CLK kinase induces neuronal differentiation of PC12 cells. Mol Cell Biol. 14 :6954-6961.
Colwill K, Pawson T, Andrews B, Prasad J, Manley JL, Bell JC, Duncan PI. (1996). The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J . 15 :265-275.
Hartmann AM, Rujescu D, Giannakouros T, Nikolakaki E, Goedert M, Mandelkow EM, Gao QS, Andreadis A, Stamm S. Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors. (2001). Mol Cell Neurosci. 18 :80-90.
Yun B, Farkas R, Lee K, Rabinow L. (1994). The Doa locus encodes a member of a new protein kinase family and is essential for eye and embryonic development in Drosophila melanogaster. Genes Dev. 8 :1160-1073.

Human cdc-2 like kinase 3 in complex with leucettine L<sub>41</sub>

Download Coordinates:

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