A R T I C L E
Backbone NMR resonance assignment of the Abelson kinase
domain in complex with imatinib
Navratna VajpaiÆ Andre´ Strauss Æ Gabriele Fendrich Æ Sandra W. Cowan-JacobÆ Paul W. Manley Æ
Wolfgang JahnkeÆ Stephan Grzesiek
Received: 26 November 2007 / Accepted: 9 January 2008 / Published online: 5 March 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Imatinib (Glivec or Gleevec) potently inhibits the tyrosine kinase activity of BCR-ABL, a constitutively activated kinase, which causes chronic myelogenous leu-kemia (CML). Here we report the first almost complete backbone assignment of c-ABL kinase domain in complex with imatinib.
Keywords Tyrosine kinases Glivec BCR-ABL Chronic myelogenous leukemia Selective labeling
Biological context
Tyrosine kinases are important mediators in signal trans-duction pathways and are tightly regulated by several mechanisms. Aberrant activation of these enzymes may lead to diseases, and mutations in kinase genes are impli-cated in many forms of cancer (Greenman et al.2007). A number of tyrosine kinases are therefore attractive drug targets for the discovery of inhibitors, which can modulate the activity of these enzymes. The BCR-ABL fusion pro-tein, having a constitutively activated Abelson (ABL)
kinase domain, is one such important drug target, which has been clinically validated by imatinib (Glivec; Novartis Pharma AG), an efficacious and well-tolerated treatment for chronic myelogenous leukemia (CML) (Ren 2005). Much structural knowledge on ABL-inhibitor complexes has been generated using X-ray crystallography (Nagar et al. 2002; Cowan-Jacob et al. 2007), but very little is known about the solution structure and dynamics of ABL kinase.
To enable NMR studies on the solution behavior of ABL kinase, we have obtained backbone resonance assignment of ABL kinase domain in complex with imatinib. The selected construct of the ABL kinase domain (denoted here as ABL, residues GAMDP-S229-S500) contains 277 resi-dues and has a typical kinase bilobal structure, with the N-terminal lobe containing b-sheets and the conserved helix C, and the C-terminal lobe being mainly helical. At the interface of the two lobes is the ATP-binding pocket, which is also the binding site of most ABL inhibitors.
Methods and materials
Since ABL is not readily expressed in E. coli, we have developed a protocol for the13C/15N isotope labeling of the ABL kinase catalytic domain in Baculovirus-infected insect cells, either uniformly or selectively for certain amino 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 in 250 ll (Shigemi microtubes) of buffer containing 95% H2O, 5% D2O, 20 mM BisTris, 100 mM NaCl, 2 mM EDTA, 3 mM DTT at pH 6.5 with an ABL:imatinib ratio of 1:1. NMR spectra were recorded at 293 K on Bruker
Electronic supplementary material The online version of this
article (doi:10.1007/s12104-008-9079-7) contains supplementary material, which is available to authorized users.
N. Vajpai S. Grzesiek (&)
Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, Switzerland
e-mail: stephan.grzesiek@unibas.ch
A. Strauss G. Fendrich S. W. Cowan-Jacob
P. W. Manley W. Jahnke (&)
Novartis Institutes for Biomedical Research, Basel, Switzerland e-mail: wolfgang.jahnke@novartis.com
123
Biomol NMR Assign (2008) 2:41–42 DOI 10.1007/s12104-008-9079-7
DRX 600 (cryo or room temperature probe) or DRX 800 MHz (cryoprobe) spectrometers. Due to the lack of deuteration, the sensitivity of NMR experiments involving 13Cb 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-1H NOESY. The assignment was aided and verified by spectra of a total of 15 selectively labeled samples that had15N, 13Ca and 13C’ nuclei introduced specifically for certain amino acid types (see supplementary material Table 1). All NMR data were processed and analysed using the NMR-Pipe (Delaglio et al. 1995) and NMRView (Johnson and Blevins1994) software suites.1H, 15N and 13C are refer-enced relative to the frequency of the2H lock resonance of water.
Extent of assignment and data deposition
The achieved assignments comprise 96% of all backbone 1HN,15N, 13Caand13CO resonances, covering 254 of the
264 non-proline residues (Fig. 1). Unassigned residues consist of the N-terminal glycine, seven residues within the activation loop (consisting of residues D381-P402), and H361. Line broadening of adjacent residues indicates that most of the missing residues are broadened beyond detection due to intermediate conformational exchange. Despite the difficulty in observing the entire activation loop, the assignments include at least 14 key residues involved in ligand binding. The assignments have been deposited in the BMRB (accession number 15488).
Acknowledgements We would like to thank Drs. Sonja Alexandra
Dames and Martin Allan for their help during the initial phase of the project. This work was supported by SNF grant 31-109712 (S.G.) and Novartis Pharma AG.
References
Cowan-Jacob SW, Fendrich G, Floersheimer A, Furet P, Liebetanz J, Rummel G, Rheinberger P, Centeleghe M, Fabbro D, Manley PW (2007) Structural biology contributions to the discovery of drugs to treat chronic myelogenous leukaemia. Acta Crystallogr D Biol Crystallogr 63:80–93
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe – a multidimensional spectral processing system based on unix pipes. J Biomol NMR 6:277–293
Greenman C, Stephens P, Smith R et al (2007) Patterns of somatic mutation in human cancer genomes. Nature 446:153–158 Grzesiek S, Bax A (1992) Improved 3D triple-resonance NMR
techniques applied to a 31-kDa protein. J Magn Reson 96: 432–440
Johnson BA, Blevins RA (1994) NMR View – a computer-program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614
Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach D R, Miller WT, Clarkson B, Kuriyan J (2002) Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res 62:4236–4243
Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371
Ren R (2005) Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer 5:172–183 Strauss A, Bitsch F, Cutting B, Fendrich G, Graff P, Liebetanz J,
Zurini M, Jahnke W (2003) Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies. J Biomol NMR 26:367–372 Strauss A, Bitsch F, Fendrich G, Graff P, Knecht R, Meyhack B,
Jahnke W (2005) Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J Biomol NMR 31:343–349 6.0 7.0 8.0 9.0 10.0 11.0 G259 G463 G249 R362 N479 E292 Y413 W476 Y264 M458K291 I293 K415 N368 L266 V448 A365 Y353 L411 L471 L376 V339 T389 S385 I360 Y320 K419 S265 R328 C475D381 K357 T392 T319 L340 C305 I313 E431 F425 I418 L370 T243 L428 W478 T267 L445 K274 H295 D444 I314 M244 R239 M237 Q300 Y257 L302 L364 E466 E255K262 A407 R332 K271 W261 A366 D363 T306 N297 F486 E409 S417 T315 D421 K294 A380 L301 G321 G251 G303 T240 G436 G398 G426 G250 K375 V304 G390 G383 G442 S446 S485 S481 G254 T277 R473 D455 M451 C369 H246 N358 E329 T495 E308 V468 V338 S410 R457 A350 S348 Y253 V270 L248 A399 F283 G372 K467 Y312 N374 Q477 T406 E316 Y440 R483 E334 M437 I443 W423 M472 M496 V427 Q447 E488E499 Q452 S349 L341 L451D482 L354 V289 V371 A344 K400 W235 M388 Q498 E373 F497 E494 K378 D391 K234 E279 L452 W405 E355 L429 L324 V268 R367 102 106 110 114 118 122 126 130 7 Q346 V299 E286 L298 Y469 W430F382 S420 M278 E275 A225 K247 K245 A487 F416 V379 V335 I489 A269 E352 F311 L273 D233 A492 A424 R307 E258 E462 E238 A4744 Y456Y232D241 S500 V256 F317 E453 V280 K285 K356 E470 E282 Y435 A412 D425 A433 N322 F359 M290 I347 N414 L284 S229 S438 N231 M226 I432 K263 Q491 V V260 V377 R360 Y342 a a c c b b 1HN ppm 15N M343 ε ε εε
Fig. 1 1HN-15N HSQC-TROSY (Pervushin et al.1997) spectrum of
uniformly 15N-labeled ABL kinase domain-imatinib complex with
assignment information. An ‘e’ denotes unassigned WeNH sidechain
resonances, Subpanels a, b and c show enlarged regions of the respective rectangular boxes in the main spectrum
42 N. Vajpai et al.
