Imatinib (Glivec or Gleevec) potently inhibits the tyrosine kinase activity of BCR-ABL, a constitutively activated kinase, which causes chronic myelogenous leukemia (CML). Here we report the first almost complete backbone assignment of c-ABL kinase domain in complex with imatini

Cowan-Jacob, Sandra

Fendrich, Gabriele

Grzesiek, Stephan

Jahnke, Wolfgang

Manley, Paul

Strauss, André

Vajpai, Navratna

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ARTICLEBackbone NMR resonance assignment of the Abelson kinasedomain in complex with imatinibNavratna Vajpai Æ Andre´ Strauss Æ Gabriele Fendrich ÆSandra W. Cowan-Jacob Æ Paul W. Manley ÆWolfgang Jahnke Æ Stephan GrzesiekReceived: 26 November 2007 / Accepted: 9 January 2008 / Published online: 5 March 2008 Springer Science+Business Media B.V. 2008Abstract Imatinib (Glivec or Gleevec) potently inhibitsthe tyrosine kinase activity of BCR-ABL, a constitutivelyactivated kinase, which causes chronic myelogenous leu-kemia (CML). Here we report the first almost completebackbone assignment of c-ABL kinase domain in complexwith imatinib.Keywords Tyrosine kinases  Glivec  BCR-ABL Chronic myelogenous leukemia  Selective labelingBiological contextTyrosine kinases are important mediators in signal trans-duction pathways and are tightly regulated by severalmechanisms. Aberrant activation of these enzymes maylead to diseases, and mutations in kinase genes are impli-cated in many forms of cancer (Greenman et al. 2007). Anumber of tyrosine kinases are therefore attractive drugtargets for the discovery of inhibitors, which can modulatethe activity of these enzymes. The BCR-ABL fusion pro-tein, having a constitutively activated Abelson (ABL)kinase domain, is one such important drug target, whichhas been clinically validated by imatinib (Glivec; NovartisPharma AG), an efficacious and well-tolerated treatmentfor chronic myelogenous leukemia (CML) (Ren 2005).Much structural knowledge on ABL-inhibitor complexeshas been generated using X-ray crystallography (Nagaret al. 2002; Cowan-Jacob et al. 2007), but very little isknown about the solution structure and dynamics of ABLkinase.To enable NMR studies on the solution behavior of ABLkinase, we have obtained backbone resonance assignmentof ABL kinase domain in complex with imatinib. Theselected construct of the ABL kinase domain (denoted hereas ABL, residues GAMDP-S229-S500) contains 277 resi-dues and has a typical kinase bilobal structure, with theN-terminal lobe containing b-sheets and the conservedhelix C, and the C-terminal lobe being mainly helical. Atthe interface of the two lobes is the ATP-binding pocket,which is also the binding site of most ABL inhibitors.Methods and materialsSince ABL is not readily expressed in E. coli, we havedeveloped a protocol for the 13C/15N isotope labeling of theABL kinase catalytic domain in Baculovirus-infectedinsect cells, either uniformly or selectively for certainamino acid types (Strauss et al. 2003; Strauss et al. 2005).Uniformly 13C,15N or selectively labeled samples(Strauss et al. 2003; Strauss et al. 2005) of the ABL-imatinib complex were prepared as 0.4 mM solutions in250 ll (Shigemi microtubes) of buffer containing 95%H2O, 5% D2O, 20 mM BisTris, 100 mM NaCl, 2 mMEDTA, 3 mM DTT at pH 6.5 with an ABL:imatinib ratioof 1:1. NMR spectra were recorded at 293 K on BrukerElectronic supplementary material The online version of thisarticle (doi:10.1007/s12104-008-9079-7) contains supplementarymaterial, which is available to authorized users.N. Vajpai  S. Grzesiek (&)Biozentrum, University of Basel, Klingelbergstrasse 70,Basel, Switzerlande-mail: stephan.grzesiek@unibas.chA. Strauss  G. Fendrich  S. W. Cowan-Jacob P. W. Manley  W. Jahnke (&)Novartis Institutes for Biomedical Research, Basel, Switzerlande-mail: wolfgang.jahnke@novartis.com123Biomol NMR Assign (2008) 2:41–42DOI 10.1007/s12104-008-9079-7DRX 600 (cryo or room temperature probe) or DRX800 MHz (cryoprobe) spectrometers. Due to the lack ofdeuteration, the sensitivity of NMR experiments involving13Cb or other side chain nuclei was very low. Therefore,backbone assignments had to be performed with non-TROSY versions of the HNCO, HNCA, HN(CO)CA(Grzesiek and Bax 1992), and an 15N-edited 1H-1HNOESY. The assignment was aided and verified by spectraof a total of 15 selectively labeled samples that had 15N,13Ca and 13C’ nuclei introduced specifically for certainamino acid types (see supplementary material Table 1). AllNMR data were processed and analysed using the NMR-Pipe (Delaglio et al. 1995) and NMRView (Johnson andBlevins 1994) software suites. 1H, 15N and 13C are refer-enced relative to the frequency of the 2H lock resonance ofwater.Extent of assignment and data depositionThe achieved assignments comprise 96% of all backbone1HN, 15N, 13Ca and 13CO resonances, covering 254 of the264 non-proline residues (Fig. 1). Unassigned residuesconsist of the N-terminal glycine, seven residues within theactivation loop (consisting of residues D381-P402), andH361. Line broadening of adjacent residues indicates thatmost of the missing residues are broadened beyonddetection due to intermediate conformational exchange.Despite the difficulty in observing the entire activationloop, the assignments include at least 14 key residuesinvolved in ligand binding. The assignments have beendeposited in the BMRB (accession number 15488).Acknowledgements We would like to thank Drs. Sonja AlexandraDames and Martin Allan for their help during the initial phase of theproject. This work was supported by SNF grant 31-109712 (S.G.) andNovartis Pharma AG.ReferencesCowan-Jacob SW, Fendrich G, Floersheimer A, Furet P, Liebetanz J,Rummel G, Rheinberger P, Centeleghe M, Fabbro D, ManleyPW (2007) Structural biology contributions to the discovery ofdrugs to treat chronic myelogenous leukaemia. Acta CrystallogrD Biol Crystallogr 63:80–93Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995)NMRPipe – a multidimensional spectral processing systembased on unix pipes. J Biomol NMR 6:277–293Greenman C, Stephens P, Smith R et al (2007) Patterns of somaticmutation in human cancer genomes. Nature 446:153–158Grzesiek S, Bax A (1992) Improved 3D triple-resonance NMRtechniques applied to a 31-kDa protein. J Magn Reson 96:432–440Johnson BA, Blevins RA (1994) NMR View – a computer-programfor the visualization and analysis of NMR data. J Biomol NMR4:603–614Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach D R, MillerWT, Clarkson B, Kuriyan J (2002) Crystal structures of thekinase domain of c-Abl in complex with the small moleculeinhibitors PD173955 and imatinib (STI-571). Cancer Res62:4236–4243Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2relaxation by mutual cancellation of dipole-dipole coupling andchemical shift anisotropy indicates an avenue to NMR structuresof very large biological macromolecules in solution. Proc NatlAcad Sci USA 94:12366–12371Ren R (2005) Mechanisms of BCR-ABL in the pathogenesis ofchronic myelogenous leukaemia. Nat Rev Cancer 5:172–183Strauss A, Bitsch F, Cutting B, Fendrich G, Graff P, Liebetanz J,Zurini M, Jahnke W (2003) Amino-acid-type selective isotopelabeling of proteins expressed in Baculovirus-infected insectcells useful for NMR studies. J Biomol NMR 26:367–372Strauss A, Bitsch F, Fendrich G, Graff P, Knecht R, Meyhack B,Jahnke W (2005) Efficient uniform isotope labeling of Ablkinase expressed in Baculovirus-infected insect cells. J BiomolNMR 31:343–3496.07.08.09.010.011.0G259 G463G249R362N479E292Y413W476 Y264M458 K291I293K415N368L266V448A365Y353L411L471L376V339T389S385I360Y320K419S265R328C475 D381K357T392T319L340C305I313E431F425I418L370T243L428W478T267 L445 K274H295D444I314M244R239M237Q300Y257L302L364E466E255 K262 A407R332K271W261A366D363T306N297F486E409S417 T315D421K294A380L301G321G251G303T240G436G398G426G250K375V304G390G383G442S446S485S481G254T277 R473D455 M451C369H246N358E329T495E308V468V338S410R457A350S348Y253V270L248A399F283G372K467Y312N374Q477T406E316Y440R483E334M437I443W423M472M496V427Q447E488E499 Q452S349L341L451D482L354V289V371A344K400W235M388Q498E373F497E494K378D391K234E279L452W405E355L429L324V268R3671021061101141181221261307Q346V299E286L298Y469W430F382S420 M278E275A225K247K245A487F416V379V335I489 A269E352F311L273D233A492A424R307E258E462E238A474 4Y456Y232D241S500V256F317E453 V280K285K356E470 E282Y435A412D425A433N322F359M290I347N414L284S229S438N231M226I432 K263Q491VV260V377R360Y342aaccbb1HNppm15NM343ε εεεFig. 1 1HN-15N HSQC-TROSY (Pervushin et al. 1997) spectrum ofuniformly 15N-labeled ABL kinase domain-imatinib complex withassignment information. An ‘e’ denotes unassigned We NH sidechainresonances, Subpanels a, b and c show enlarged regions of therespective rectangular boxes in the main spectrum42 N. Vajpai et al.123

Backbone NMR resonance assignment of the Abelson kinase domain in complex with imatinib

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Backbone NMR resonance assignment of the Abelson kinase domain in complex with imatinib

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