Crystal Structure of Human cdc2-like Kinase (CLK1) in Complex with Debromo-hymenialdisine

Alex N. Bullock1, Santina Russo2, Ann Amos1, Nicholas Pagano3, Howard Bregman4,Judit É. Debreczeni1, Wen Hwa Lee1, Eric Meggers3, Stefan Knapp1*

1 University of Oxford,Structural Genomics Consortium, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.

2 Swiss Light Source. Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.

3 Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany.

4 Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA. New address: Amgen Inc., 1 Kendall Square, Cambridge, MA 02139, USA.

* Corresponding author e-mail: stefan.knapp@sgc.ox.ac.uk

PDB Code: 2IWI (deposited on 30.Jun.06)

Datapack version: 1 (built on 12.Jan.09; last revised on 12.Jan.09)

Abstract

The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2. Here we determined the crystal structure of PIM2 in complex with a ruthenium half-sandwich complex. Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases. The structure of PIM2 kinase reveals several differences to PIM1 which may be explored further to generate isoform selective inhibitors.

Introduction

The Clk family consists of four kinases that display so-called dualspecificity, i.e., they are capable of phosphorylating both Ser/Thr aswell as Tyr residues.

CLK homologues have been isolated from a number of organismsincluding Arabidopsis, Drosophila, mouse and human. The kinase domainof CLK family members is located at the C terminus and contains thefamily signature motif ‘EHLAMMERILG’, giving rise to the name of thesekinases: ‘LAMMER’ kinases. Mutations in the Drosophila CLK homologue,darkener of apricot (DOA) suggest that CLK family members play animportant role during development. Mutations in DOA are embryoniclethal and lead to defects in differentiation, including abnormalitiesin segmentation, eye formation, and neuronal development.

CLK1 also plays an important role regulating RNA splicing. It hasbeen shown that CLK1 forms complexes with and phosphorylates members ofthe SR family of splicing factors. Overexpression affects splicing siteselection of pre-mRNA of both its own transcript and adenovirus E1Atranscripts in vivo which led to the current model that Clk familymembers regulate alternative splicing by phosphorylation of SRproteins. In addition, overexpression of catalytically inactive CLK1localize to nuclear speckles where splicing factors are concentrated,whereas the wild-type enzymes distribute throughout the nucleus andcause speckles to dissolve.

We solved the structure of human CLK1 kinase domain at 1.7 Åresolution in complex with the marine sponge metabolite hymenialdisinewhich has been shown to be a potent ATP-competitive inhibitor of MEK-1,Cdk’s, GSK-3b and CK1. Hymenialdisine also inhibits G2 phase DNA damagecheckpoint and blocks IL-8 production in U937 cells by inhibiting theactivation of NF-kB. Hymenialdisine is therefore a potentialtherapeutic reagent for the treatment of diabetes and neurodegenerativedisorders

Results

Overall structure: The amino-terminal lobe of CLK1 has a secondary structuretypical for kinases which usually comprising three β-sheets followed byhelix α C and two β-sheets. The most striking feature observed in theC-terminal lobe is an insertion between the sheets β7 and β10 (residues S299-I317 in CLK1). This insertion is conserved withinthe CLK family but has however only low sequence homology when comparedwith other CLK family members. The insertion forms a β-hairpinstructure (β8 and β9) that packs against a groove formed by helix αDand αE.

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The conserved CLK signature motif 'EHLAMMERILG' (residues 386-396 in CLK1), which gave rise to the family name'LAMMER' kinases, is part of helix αG. This sequence stretch is part ofthe lower lobe core structure and is largely inaccessible.

An interesting feature of the low lobe structure became evidentcomparing CLK1 with kinases of the MAPK family. MAP kinases show a characteristic insertion of a helix-loop-helix motif (human P38 in orange; human CLK1 in red) in the C-terminal lobe.The first helix is formed by CLK1 residues K400 to T406. However, theCLK1 residues Arg409-Asp418 from a coil-sheet structure that followsthe second helix in P38 in structural alignments. In addition, residuesSer424-Ala432 form a small helix termed αJ unique to CLK1 (with P38 shown in yellow for comparison).

Active site residues: Comparison with other kinase structures showed that conserved residuesimportant for catalysis and cofactor binding are correctly oriented andform interactions typical for active kinases. Like the conserved Lys191 which coordinates phosphates in ATP forms a salt bridge with Glu206 in helix αC . The DFG-motif (CLK1 residues 325-327), which usually coordinates Mg2+ ions bound to the nucleotide, as well as the catalytic base Asp288 has an identical side chain orientation as in active Aurora-A (pdb-code 1OL7).

Activation loop residues: The entire CLK1 activation loop is well ordered and has a low overall B factor. The conformationof the loop is similar to conformations observed in active kinases.Interestingly, Tyr331 forms a hydrogen bond with αC residue His212. Polar interaction formed by activation loop phosphate moieties andresidues in αC is a hallmark of active kinases and is thought tostabilize activation loop conformation. It is therefore probable thatTyr331 mimics such an interaction and stabilizes loop conformation.However, tyrosine at that position is only present in CLK1 and CLK4 butnot in the other family members CLK2 and CLK3. Several polar activationloop side-chains stabilize the ordered conformation of the loop byforming hydrogen bonds with main chain atoms. Like His212 forms a hydrogen bond with the main chain Tyr331 oxygen, Glu334 with main chain Tyr334 and His 336 with carbonyl oxygen of its neighbour His335. However, the CLK1 activation is involved in crystal contacts and theobserved conformation and stability might be as well influenced byneighbouring protein molecules. In addition, binding of the inhibitorhyemandisine has been reported to stabilize activation loopconformation forming a hydrogen bond with the DFG aspartate.

Binding of debromo-hymenialdisine: The inhibitor debromo-hymenialdisine forms several direct hydrogen bonds with key elements of kinaseactive site explaining why this inhibitor has been reported to inhibitseveral protein kinases. Two direct hydrogen bonds and one watermediated hydrogen bond were observed with the hinge backbone formed bythe CLK1 residues Glu242, Leu243 and Leu244. Another hydrogen bond isformed between the imidazolone carbonyl oxygen and the conserved lysineLys191 as well as the amino group with the aspartate in the activationloop DFG motif Asp305. In addition, this amino group forms a hydrogenbond with Asn294. All in all the binding mode of debromo-hyemandisineobserved in the CLK1 co-crystal structure is conserved and resemblesinteractions observed in the CDK2 and ACK1 complex.

Debromo-hymenialdisine

Discussion

The proto-oncogene PIM2 is a key mediator of hematopoietic cell growth and apoptotic resistance and complements transformation by c-MYC and mutant tyrosine kinases including BCR/ABL and FLT3-ITD. Importantly, PIM2 inactivation can restore apoptosis to otherwise drug-resistant cancers and is therefore an attractive therapy to supplement current drug regimes such as Gleevec™. The structure of PIM2 reveals a constitutively active conformation consistent with the view that PIM2 activity is regulated principally at the transcriptional level [11]. Consequently, the oncogenic potential of PIM2 is greatly increased on overexpression.

Overall, the structure is highly similar to PIM1, particularly in the ATP pocket which is nearly completely conserved in comparison to the overall sequence identity of 55%. The generally reduced susceptibility of PIM2 to previously characterized PIM1 inhibitors such as LY331'531 [28] might instead result from a change in protein dynamics as suggested here by several disordered loops in the N-terminal kinase lobe. The main structural distinction between the two kinases is the absence of the αJ helix in PIM2 which removes a significant stabilizing interaction close to the interface between the N and C-terminal lobes as well as differences in the kinase hinge and P loop residues.

Based on the initial staurosporine scaffold the ruthenium half-sandwich complexes have provided marked specificity for the GSK3 and PIM kinases by the introduction of the metal centre coordinated by a cyclopentadienyl ring and a CO ligand [23,25,26]. The structures of PIM1 and now PIM2 bound to 1 show a remarkable fit between the inhibitor and the ATP pocket that explains the inhibitor’s potency. Our SAR analysis highlights the promise for further scaffold optimization with both kinases having particular preference for a hydroxyl substituent at the R1 position (compound 2) [27,29]. The structure of PIM1 in complex with compound 2 showed similar positions for the maleimide group, the cyclopentadienyl ring and the CO ligand, but a 180° flip in the pyridocarbazole moiety that enables two water-mediated hydrogen bonds to form through the R1 hydroxyl with Glu89 [23].

Acknowledgements

The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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