Structure of the Epidermal Growth Factor Receptor Kinase Domain Alone and in Complex with a 4-Anilinoquinazoline Inhibitor

The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-Å resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, TarcevaTM). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization. The crystal structure of the kinase domain from the epidermal growth factor receptor (EGFRK) including forty amino acids from the carboxyl-terminal tail has been determined to 2.6-Å resolution, both with and without an EGFRK-specific inhibitor currently in Phase III clinical trials as an anti-cancer agent, erlotinib (OSI-774, CP-358,774, TarcevaTM). The EGFR family members are distinguished from all other known receptor tyrosine kinases in possessing constitutive kinase activity without a phosphorylation event within their kinase domains. Despite its lack of phosphorylation, we find that the EGFRK activation loop adopts a conformation similar to that of the phosphorylated active form of the kinase domain from the insulin receptor. Surprisingly, key residues of a putative dimerization motif lying between the EGFRK domain and carboxyl-terminal substrate docking sites are found in close contact with the kinase domain. Significant intermolecular contacts involving the carboxyl-terminal tail are discussed with respect to receptor oligomerization. Growth factor interactions with cell surface receptors influence proliferation, survival, differentiation, and metabolism (1Schlessinger J. Ullrich A. Neuron. 1992; 9: 383-391Abstract Full Text PDF PubMed Scopus (1292) Google Scholar). The loss of control over these vital cellular processes is a hallmark of oncogenesis (2Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar). For instance, aberrant signaling from overexpressed growth factor receptor ErbB2 is causal in approximately 30% of invasive breast cancers (3Ross J.S. Fletcher J.A. Stem Cells. 1998; 16: 413-428Crossref PubMed Scopus (614) Google Scholar). Growth factors bind to a cognate membrane-bound receptor system and mediate changes in the intracellular portion of the receptor, often through the formation of dimers or oligomers of receptors that initiate signal transduction cascades. The epidermal growth factor receptor (EGFR, 1The abbreviations used for: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; RTK, receptor tyrosine kinase; SH2, Src homology 2; p-Tyr, phosphotyrosine; A-loop, activation loop; DTT, dithiothreitol; P38, mitogen-activated protein kinase p38; MES, 4-morpholineethanesulfonic acid; FGFRK, fibroblast growth factor receptor kinase; EGFRK, epidermal growth factor receptor kinase; N-lobe, NH2-terminal lobe; C-lobe, COOH-terminal lobe; r.m.s., root mean square; LCK, lymphocyte tyrosine kinase; p-IRK, insulin receptor kinase-phosphorylated form; FGF, fibroblast growth factor; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; CDK2, cyclin-dependent kinase 2. also ErbB1 or HER1) and its ligands, epidermal growth factor (EGF) and transforming growth factor-α, are among the earliest characterized members of the growth factor/receptor tyrosine kinase (RTK) family. In contrast to the widely applicable ligand-induced receptor dimerization paradigm, there is evidence that EGFR family members exist as preformed dimers (4Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar) and form higher oligomer signaling complexes (5Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3538) Google Scholar). Normal signaling in the EGFR system involves ligand-induced homo-oligomerization or hetero-oligomerization with the closely related RTKs ErbB2 (HER2), ErbB3 (HER3) and/or ErbB4 (HER4) (6Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5633) Google Scholar). Autophosphorylation of key tyrosine residues within the carboxyl-terminal portion of the receptor provides sites for direct interaction with SH2-containing proteins, leading to subsequent signal transduction events. The EGFR system, including receptor homologues and relevant ligands, is complex. There are at least 12 different ligands that bind to the EGF receptor family with partially redundant specificity for certain receptors. Several of the ligands including EGF, transforming growth factor-α, heparin-binding EGF, and betacellulin are reported to bind to EGFR with nanomolar dissociation constants (7Jones J.T. Akita R.W. Sliwkowski M.X. FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (320) Google Scholar). Betacellulin also binds ErbB4 with high affinity. Similarly, heregulin binds to ErbB3 or ErbB4 with dissociation constants in the nanomolar range. So far, a ligand that binds ErbB2 alone has not been identified, although the affinity of an ErbB2/ErbB3 heterodimer for heregulin is high, ∼1011m (8Karunagaran D. Tzahar E. Beerli R.R. Chen X. Graus-Porta D. Ratzkin B.J. Seger R. Hynes N.E. Yarden Y. EMBO J. 1996; 15: 254-264Crossref PubMed Scopus (588) Google Scholar, 9Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway III., K.L. J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar). In addition, the kinase domain of ErbB3 has non-canonical amino acids at some key positions, which render it catalytically inactive (10Guy P.M. Platko J.V. Cantley L.C. Cerione R.A. Carraway III., K.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8132-8136Crossref PubMed Scopus (596) Google Scholar). Taken together, these factors point to a complicated interplay between cross-reacting ligands, functional diversity among receptors, and differential expression in the EGFR signaling system. In the non-signaling state, most RTKs possess low basal kinase activity that increases substantially upon growth factor binding. This results from receptor oligomerization and subsequent transphosphorylation of tyrosine residues within a partner kinase domain. Specifically, initial phosphotyrosine (p-Tyr) modification of the "activation loop" (A-loop) generates optimal catalytic activity and subsequent rapid phosphorylation at substrate docking sites elsewhere on the receptor intracellular domain. The EGFR, ErbB2, and ErbB4 receptors are the only known RTKs that do not require this initial phosphorylation of kinase domain residues for full catalytic competency. This unique feature may partially explain why EGFR family members are frequently involved in cellular transformation. In the RTKs for which crystal structures of both unphosphorylated and phosphorylated versions of the kinase domain are available, phosphorylation in the A-loop causes it to undergo a large structural reorganization that relieves steric and/or chemical restraints on the catalytic active site (11Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (892) Google Scholar). Distinguishing the EGFR family further is an intracellular dimerization motif that has been roughly assigned to reside between the kinase domain and the carboxyl-terminal phosphorylation sites. The greatest effects on receptor function seem to be concentrated in the Leu955-Val956-Ile957 segment of EGFR ("LVI") and other ErbB receptors. This motif is necessary for ligand-independent dimerization of EGFR intracellular domains (12Chantry A. J. Biol. Chem. 1995; 270: 3068-3073Abstract Full Text Full Text PDF PubMed Google Scholar) and for transphosphorylation in ErbB2/ErbB3 heterodimers (13Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Moreover, alanine substitutions in this region override mutations in the transmembrane segment of ErbB2 that would otherwise lead to constitutive signaling via non-ligand induced dimerization (14Penuel E. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 2002; 277: 28468-28473Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The molecular mechanism by which these residues influence receptor activity is not well understood. Members of the EGFR family are frequently overactive in solid tumors (15Khazaie K. Schirrmacher V. Lichtner R.B. Cancer Metastasis Rev. 1993; 12: 255-274Crossref PubMed Scopus (267) Google Scholar). A number of therapeutic approaches that interfere with aberrant EGFR family signaling are being investigated (16Shawver L.K. Slamon D. Ullrich A. Cancer Cells. 2002; 1: 117-123Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). A relatively new therapeutic approach to kinase inhibition is the use of ATP-competitive small molecules (17Druker B.J. Sawyers C.L. Kantarjian H. Resta D.J. Reese S.F. Ford J.M. Capdeville R. Talpaz M. N. Eng. J. Med. 2001; 344: 1038-1042Crossref PubMed Scopus (2442) Google Scholar, 18Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1621) Google Scholar, 19Arteaga C.L. J. Clin. Oncol. 2001; 19: S32-S40PubMed Google Scholar, 20Kelloff G.J. Fay J.R. Steele V.E. Lubet R.A. Boone C.W. Crowell J.A. Sigman C.C. Cancer Epidemiol. Biomark. Prev. 1996; 5: 657-666PubMed Google Scholar). Several groups have shown that certain 4-anilinoquinazoline derivatives are both selective and effective inhibitors of the EGFR kinase (21Woodburn J.R. Pharmacol. Ther. 1999; 82: 241-250Crossref PubMed Scopus (780) Google Scholar). Structural data exist for compounds of this general class bound to the distantly related intracellular kinases CDK2 and mitogen-activated protein kinase p38 (P38) (22Shewchuk L. Hassell A. Wisely B. Rocque W. Holmes W. Veal J. Kuyper L.F. J. Med. Chem. 2000; 43: 133-138Crossref PubMed Scopus (207) Google Scholar, 23.Deleted in proof.Google Scholar, 24.Deleted in proof.Google Scholar). Many of these inhibitors are being tested for the treatment of cancer including erlotinib (OSI-774, CP-358,774, TarcevaTM), which is currently undergoing Phase III clinical study. Despite extensive study of the EGFR family, only very recently have molecular structures been determined for any fragment. Crystal structures of extracellular domains from EGFR (47Osigo H. Ishitani R. Nureki O. Fukai S. Yamanaka M. Kim J.-H. Saito K. Sakamoto A. Inoue M. Shirouzu M. Yokohama S. Cell. 2002; 110: 775-787Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 48Garrett T.P.J. McKern N.M. Lou M. Elleman T.C. Adams T.E. Lovrecz G.O. Zhu H.-J. Walker F. Frenkel M.J. Hoyne P.A. Jorissen R. Nice E.C. Burgess A.W. Ward C.W. Cell. 2002; 110: 763-773Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar) and HER3 (49Cho H.-S. Leahy D.J. Science. 2002; 297: 1330-1333Crossref PubMed Scopus (351) Google Scholar) have been reported. X-ray crystal structures of kinase domains of several RTKs have been reported, although unlike EGFRK, all of these kinases require phosphorylation for full activity. Previous computational studies have suggested possible binding modes for the 4-anilinoquinazoline class of inhibitors to the EGFR kinase domain (25Palmer B.D. Trumppkallmeyer S. Fry D.W. Nelson J.M. Showalter H.D.H. Denny W.A. J. Med. Chem. 1997; 40: 1519-1529Crossref PubMed Scopus (148) Google Scholar,26Wissner A. Berger D.M. Boschelli D.H. Floyd M.B. Greenberger L.M. Gruber B.C. Johnson B.D. Mamuya N. Nilakantan R. Reich M.F. Shen R. Tsou H.R. Upeslacis E. Wang Y.F. Wu B.Q. Ye F. Zhang N. J. Med. Chem. 2000; 43: 3244-3256Crossref PubMed Scopus (195) Google Scholar), but no direct structural evidence has been generated thus far. Here we present the crystallographic analysis of the EGFR kinase alone and in complex with the inhibitor erlotinib. DNA encoding residues 672–998 was amplified from full-length EGFR cDNA (27Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Nature. 1984; 309: 418-425Crossref PubMed Scopus (1992) Google Scholar) by PCR, incorporating an NH2-terminal NheI restriction site and a COOH-terminal stop codon and XhoI site. The product was digested with NheI and XhoI and ligated into the appropriately digested pET-28b (Novagen, Madison, WI). Further PCR was performed on the EGFRK-pET-28b plasmid to acquire the histidine tag and thrombin site using an NH2-terminal primer with a NotI site and a COOH-terminal primer with anXbaI site. The product was digested with NotI andXbaI and ligated into similarly digested pVL1392 (BD Biosciences). Spodoptera frugiperda insect cells, SF9, were transfected with the EGFRK-pVL1392 plasmid using the Baculogold transfection system (BD Biosciences) according to the manufacturer's protocol. One liter of High FiveTM cells (Invitrogen and Expression Systems, Woodland, CA) in suspension at 5 × 105 cells/ml was inoculated with 8 ml of amplified EGFRK virus and incubated at 27 °C for 72 h. Cells were harvested by centrifugation at 4000 ×g for 15 min. Cells were frozen on dry ice and then thawed twice. 150 ml of buffer (50 mm Tris, pH 7.5, 200 mm NaCl, 1% glycerol, 1 mm DTT, 0.1 mm benzamidine) was added to the cells. The cells were then mechanically homogenized. The lysate was centrifuged at 25000 ×g for 45 min to remove insoluble material. The supernatant was passed over a 0.45-μm vacuum filter with pre-filter. Filtrate was loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen). The column was then washed with buffer (50 mm Tris, pH 8.0, 500 mm NaCl, 5 mm imidazole) for 10-column volumes. EGFRK protein was eluted from the column with 4 × 1-column volume aliquots of elution buffer (50 mm Tris, pH 8.0, 300 mm NaCl, 250 mm imidazole). Fractions containing EGFRK as assayed by SDS-PAGE were pooled, combined with thrombin to remove the histidine tag, and then dialyzed against 50 mm Tris, pH 8.0, 250 mm NaCl, 1 mmDTT. EGFRK was then concentrated to a volume of 500 μl and loaded onto a Superdex 75 gel filtration column (Amersham Biosciences) pre-equilibrated with 50 mm Tris, pH 8.0, 500 mm NaCl, 1 mm DTT. Fractions containing EGFRK as assayed by SDS-PAGE were pooled and dialyzed against 10 mm Tris, pH 8.0, 1 mm DTT, 1 mmsodium azide, 0.1 mm benzamidine. Mass spectrometry confirmed that the protein lacked any phosphorylation. The EGFRK was then concentrated to ∼8 mg/ml. Typical final yield for each liter of High Five culture was 1–2 mg. Small crystals of EGFRK formed over 1 day in hanging drops when protein was mixed with the reservoir buffer (1.0 m Na/K tartrate, 0.1 m MES, pH 7.0) in a 1:1 ratio. These crystals were used as macro seeds in a 10 μl of sitting drop containing a 1:1 protein:reservoir ratio described as above. Crystals grew to ∼250 μm3 in 1 week. Crystals of EGFRK complexed with erlotinib were obtained by soaking crystals of apo-EGFRK in a solution containing 1.1 m Na/K tartrate, 0.1m MES, pH 7.0, 3 μm erlotinib, for 3 weeks. Crystals with and without the erlotinib treatment were immersed in a reservoir solution with added glycerol (20%) before preservation with liquid nitrogen. Diffraction data were collected at beamline 19-ID of the Structural Biology Center (Advanced Photon Source, Argonne National Laboratory) and at beamline 5.0.1 of the Berkeley Center for Structural Biology (Advanced Light Source, Lawrence Berkeley National Laboratory), extending to 2.6 Å for both apo-EGFRK and EGFRK/erlotinib crystals, respectively (TableI). Data were reduced with HKL2000 and Denzo/Scalepack (28Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38573) Google Scholar). The high symmetry of the crystals affords very high redundancy in a 90°-sweep. We chose the high resolution limit of data used in refinement based on a signal-to-noise criterion (I/ς(I) ≥ 2) rather than on agreement factors.Table IStructure statistics for EGFRK/erlotinib and apo-EGFRKData collection and reductionResolution (Å)N measaNmeas is the total number of observations measured.N refbNref is the number of unique reflections measured at least once.CompletecComplete is the percentage of possible reflections actually measured at least once.I/ςRmergedRmerge = Σ‖ ‖I‖ − ‖〈I〉‖ ‖/Σ‖〈I〉‖ where I is the intensity of a single observation and 〈I〉 the average intensity for symmetry equivalent observations. The value for the highest resolution shell of EGFRK/erlotinib is not reported by Scalepack.RworkeRwork = Σ‖F o −F c‖/Σ‖F o‖ whereF o and F c are observed and calculated structure factor amplitudes, respectively.RfreefRfree = Rwork for 679 (apo-EGFRK) or 689 (EGFRK/erlotinib) reflections sequestered from refinement.EGFRK/erlotinib30.0–5.59192261744100350.0540.2680.3225.59–4.44189101663100380.0580.1790.2364.44–3.88188971664100370.0670.1920.2133.88–3.53187171674100310.0880.2180.2913.53–3.28183691656100220.1300.2240.3093.28–3.08181391663100140.2340.2670.2783.08–2.931753416261008.80.3340.3660.3712.93–2.801762916481005.60.5190.3400.4392.80–2.691775116551003.60.7920.5160.4982.69–2.601717316351002.80.5850.57730.0–2.6018234516628100270.0930.2510.294apo-EGFRK50.0–5.60281081689100640.0430.2610.3235.60–4.45294461642100800.0550.1860.2584.45–3.88294521626100660.0940.2010.2223.88–3.53294301617100430.1490.2210.2393.53–3.28295551605100250.2700.2300.3093.28–3.08301231625100140.2450.2430.3363.08–2.932368215821007.70.3460.2590.2652.93–2.801703616171004.40.4360.2660.3092.80–2.691622016121002.40.7320.4070.5012.69–2.601509315681001.80.9640.4540.55350.0–2.6024814516183100340.1080.2380.286Refinement statisticsContents of modelr.m.s. deviationsResiduesAtomsgNumbers in parenthesis is number of atoms assigned zero occupancy.WatersBondsAnglesB-factors (bonded atoms)Å°Å2EGFRK/erlotinib3142560 (37)200.0111.57.7apo-EGFRK3072469 (30)170.0101.46.2a Nmeas is the total number of observations measured.b Nref is the number of unique reflections measured at least once.c Complete is the percentage of possible reflections actually measured at least once.d Rmerge = Σ‖ ‖I‖ − ‖〈I〉‖ ‖/Σ‖〈I〉‖ where I is the intensity of a single observation and 〈I〉 the average intensity for symmetry equivalent observations. The value for the highest resolution shell of EGFRK/erlotinib is not reported by Scalepack.e Rwork = Σ‖F o −F c‖/Σ‖F o‖ whereF o and F c are observed and calculated structure factor amplitudes, respectively.f Rfree = Rwork for 679 (apo-EGFRK) or 689 (EGFRK/erlotinib) reflections sequestered from refinement.g Numbers in parenthesis is number of atoms assigned zero occupancy. Open table in a new tab Space group symmetry and cell parameters suggested 1 molecule/asymmetric unit (V m = 3.5 Å3/Dalton) and 61% solvent. The apo-EGFRK structure was solved (CCP4, Amore) (29CCP4. Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar) using a polyalanine version of FGFRK (Protein Data Bank code 1fgk) in space group I23 using data to 4.0 Å from an in-house data set (rotating anode (RU-200)/Mar345 scanner) reduced with Denzo/Scalepack. Before refinement, 4% of the data were sequestered for the calculation of Rfree, and the same set was used (and extended to higher resolution) for the synchrotron data sets. Initial phases treated with SIGMAA (30Read R.J. Acta Crystallogr. Sec. A. 1986; 42: 140-149Crossref Scopus (2036) Google Scholar) and solvent flattened (DM) produced a map with indications of many buried side chains, especially leucines and tryptophans. Model inspection and adjustment were performed with XtalView (31McRee D.E. Practical Protein Crystallography. 2nd Ed. Academic Press, Orlando, FL1999Google Scholar), and refinement employed XPLOR98 (Accelrys, San Diego, CA). The apo-EGFRK structure was used as a starting point for the EGFRK/erlotinib work, and the final stages of refinement were performed similarly. The COOH terminus of our construct lacks structural similarity to any part of the template FGFRK-starting structure. The 13-residue section immediately following His964 is too poorly ordered to be fit. Weak electron density was assigned starting with residue Leu977, which leaves a 7-Å gap for the unassigned amino acids. Alternate connectivities would bridge gaps of 22 or 29-Å. The final section of the COOH terminus is well ordered where it forms intermolecular contacts with two neighboring molecules within the crystal. The activation loop is traced for its entire length. Individual isotropic temperature factors were refined, and a bulk solvent term was included. The average temperature factors are high (∼70 Å2), and the maximum permitted B was 120 Å2 attained by 1–2% of the atoms. Final (F o − F c) electron density maps lack interpretable features. Coordinates of apo-EGFRK and EGFRK/erlotinib are available from the Protein Data Bank (50Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27555) Google Scholar) (PDB accession codes 1M14 and 1M17, respectively). The EGFR kinase domain (EGFRK) adopts the bilobate-fold characteristic of all previously reported protein kinase domains (Fig.1). The NH2-terminal lobe (N-lobe) is formed from mostly β-strands and one α-helix (αC), whereas the larger COOH-terminal lobe (C-lobe) is mostly α-helical. The two lobes are separated by a cleft similar to those in which ATP, ATP analogues, and ATP-competitive inhibitors have been found to bind. Important elements of the catalytic machinery bordering the cleft on the N-lobe include the glycine-rich nucleotide phosphate-binding loop (Gly695-Gly700), whereas the C-lobe contributes the DFG motif (Asp831-Gly833), the presumptive catalytic (general base) Asp813, the catalytic loop (Arg812-Asn818), and the A-loop (Asp831-Val852). The NH2-terminal lobe of EGFRK adopts a tertiary structure similar to previously observed structures of RTKs (r.m.s. deviations for superpositioning C-α atoms with the kinase domain from the fibroblast growth factor receptor is ∼1.2 Å), although a few features distinguish the N-lobe of EGFRK from other kinase domains. The NH2 terminus of our construct begins 25 amino acids before the first glycine of the nucleotide phosphate-binding loop and includes five additional amino acids prior to Ser671 that derive from the expression construct. The first residue we identify in the electron density maps is Gly672 with the succeeding 13 residues adopting an extended conformation. The NH2-terminal nine amino acids are influenced by several intermolecular contacts including H-bonds involving main chain atoms of residues Asn676 and Leu680, although intramolecular H-bonds between Asn676 and both Tyr740 and Ser744also contribute. At Glu685, the polypeptide chain assumes a trace more similar to those of the lymphocyte tyrosine kinase (LCK, PDB code 3lck) (32Yamaguchi H. Hendrickson W.A. Nature. 1996; 384: 484-489Crossref PubMed Scopus (423) Google Scholar), the insulin receptor kinase-phosphorylated form (33Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (784) Google Scholar) (p-IRK, PDB code 1ir3), and the unphosphorylated form of the FGF receptor kinase (34Mohammadi M. Schlessinger J. Hubbard S.R. Cell. 1996; 86: 577-587Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar) (FGFRK, PDB code 1fgk). However, EGFRK lacks the tryptophan-glutamate ("WE") motif found in these related kinases, and has Arg681-Ile682 instead. In the WE-containing kinases, hydrophobic interactions of the tryptophan and an H-bond between the glutamate and a threonine or serine and the neighboring β-strand tie the NH2-terminal region to the N-lobe. In EGFRK, Arg681 projects into solvent, but Ile682 contacts Leu782 and Ile756 on the neighboring β-strand and thereby affords a similar effect. Among the canonical features characterizing the N-lobes of active forms of kinases is a salt bridge between two highly conserved side chains that interact with the α- and β-phosphates when ATP or a close homologue is present. In both the apo-EGFRK and inhibitor-bound forms of EGFRK, we find such a salt bridge between Lys721and Glu738. Our observation of this salt bridge in apo-EGFRK indicates that EGFR does not require large rearrangements within the N-lobe for catalytic competence. The COOH-terminal domain of EGFRK contains the usual organization of α-helices present in other kinase domain structures. Superpositioning of the C-lobes of kinase domains from both LCK and p-IRK yield a r.m.s. deviation of 1.1 Å. However, as with the N-lobe, a few key features differ from previously elucidated RTK structures. In most protein kinases, the activation loop assumes its catalytically competent conformation only if it first becomes phosphorylated on a Tyr or Thr. For these kinases, the unphosphorylated activation loop is positioned many Angstroms from the active conformation and may include a direct inhibitory element. For instance, the unphosphorylated A-loop in FGFRK is incompatible with substrate binding, and the unphosphorylated insulin receptor kinase A-loop blocks ATP binding as well as the substrate tyrosine site. The A-loop in apo-EGFRK (and EGFRK/erlotinib) differs significantly from other apo-, unphosphorylated A-loop structures. Earlier work has shown that Tyr845 of the EGFRK A-loop, at a position that is phosphorylated in other RTKs, can be replaced by Phe without loss of function (35Gotoh N. Tojo A. Hino M. Yazaki Y. Shibuya M. Biochem. Biophys. Res. Commun. 1992; 186: 768-774Crossref PubMed Scopus (105) Google Scholar). Consistent with this finding, we see that the A-loop of EGFRK adopts an "active" conformation similar to the phosphorylated A-loop of p-IRK (Fig.2). Many energetically beneficial interactions stabilize this conformation, most of which are also found in other active kinase A-loops. Tyr845 aligns well structurally with p-Tyr1163 of p-IRK and makes van der Waals contact with the aliphatic part of neighboring Lys836, a residue that occupies the space of Arg1155 in the p-IRK structure. An H-bond between side chains of Tyr845 and Glu848 mimics that between p-Tyr1163 and the main chain nitrogen of Gly1166 in p-IRK, but the electron density supporting this Glu848 side chain conformation is weak (Fig.3). This interaction may be important for the loop conformation, but there is another more significant aspect of Glu848. The relationship between Tyr845 and Arg812 (preceding the catalytic Asp813) is the same as between the analogous residues in p-IRK and other tyrosine kinases. This relationship is central to arranging the catalytic machinery and substrate for phospho-transfer. In p-IRK, Tyr1163 is phosphorylated, and in EGFRK, the Glu848 carboxylate can assume a position closely analogous to that of the phosphate of p-Tyr1163 in p-IRK.Figure 3Representative electron density from the EGFRK/erlotinib structure. Map (2F o −F c contoured at 1.0 r.m.s. deviation) in part of the A-loop with additional residues Arg812 (immediately precedes the catalytic Asp813) and Arg808(H-bonds to main chain of Gly839) is shown. The placement of the Glu848 side chain is not supported by electron density but is among possible low energy conformers. Extra electron density at the Tyr845 hydroxyl suggests that it interacts with a solvent molecule, Glu848, or both. Consistent with expectations from prior studies, mass spectrometry indicates no phosphorylation on the protein. Glu848 in the conformer shown mimics the phosphate of p-Tyr1163 of the activated insulin receptor kinase (Fig. 2).View Large Image Figure ViewerDownload (PPT) Anti-parallel β-strand main chain to main chain H-bonds between Lys836-Leu838 and Val810-Arg808 are key anchors for the conformation adopted by the early part of the EGFRK A-loop, and analogous interactions appear in p-IRK. However, in an additional interaction not seen in the p-IRK structure, the side chain of Arg808 H-bonds to the main chain oxygen of Gly839. This interaction lends further stability to the unphosphorylated EGFRK A-loop active conformation, and it is noteworthy that the incidence of arginine at position 808 among kinases is low. Underlying the central part of the A-loop, Tyr867 accepts an H-bond from Arg812. Whereas many kinases have a homologous Arg preceding the catalytic Asp813, its interactions with a homologous Tyr (or sometimes Phe) vary in type. Some are purely hydrophobic, whereas others involve Arg hydrogen atoms and either the hydroxyl oxygen or π-electrons of the tyrosine. Tyr867 π-electrons interact with Arg813 in EGFRK with the details very similar to those found in p-IRK. There are other protein kinases with known molecular structures that do not require phosphorylation in their A-loop for optimal catalytic competence, among them, glycogen phosphorylase kinase (36Owen D.J. Noble M.E. Garman E.F. Papageorgiou A.C. Johnson L.N. Structure. 1995; 3: 467-482Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) (PDB code1phk), casein kinase 1 (37Xu R.M. Carmel G. Sweet R.M. Kuret J. Cheng X. EMBO J. 1995; 14: 1015-1023Crossref PubMed Scopus (181) Google Scholar) (PDB code 1csn), carboxyl-terminal Src kinase (38O