HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 48, 46265-46272, November 29, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/48/46265    most recent M207135200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stamos, J. Articles by Eigenbrot, C. Search for Related Content PubMed PubMed Citation Articles by Stamos, J. Articles by Eigenbrot, C. Social Bookmarking                      What's this?

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

Jennifer Stamos, Mark X. Sliwkowski^§, and Charles Eigenbrot^¶

From the Departments of  Protein Engineering and ^§ Molecular Oncology, Genentech, Inc., South San Francisco, California 94080

Received for publication, July 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, Tarceva^TM). 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth factor interactions with cell surface receptors influence proliferation, survival, differentiation, and metabolism (1). The loss of control over these vital cellular processes is a hallmark of oncogenesis (2). For instance, aberrant signaling from overexpressed growth factor receptor ErbB2 is causal in approximately 30% of invasive breast cancers (3). 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,¹ also ErbB1 or HER1) and its ligands, epidermal growth factor (EGF) and transforming growth factor-, 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 (4) and form higher oligomer signaling complexes (5). 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) (6). 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-, heparin-binding EGF, and betacellulin are reported to bind to EGFR with nanomolar dissociation constants (7). 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, ~10¹¹ M (8, 9). In addition, the kinase domain of ErbB3 has non-canonical amino acids at some key positions, which render it catalytically inactive (10). 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 (11).

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 (12) and for transphosphorylation in ErbB2/ErbB3 heterodimers (13). 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 (14). 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 (15). A number of therapeutic approaches that interfere with aberrant EGFR family signaling are being investigated (16). A relatively new therapeutic approach to kinase inhibition is the use of ATP-competitive small molecules (17-20). Several groups have shown that certain 4-anilinoquinazoline derivatives are both selective and effective inhibitors of the EGFR kinase (21). Structural data exist for compounds of this general class bound to the distantly related intracellular kinases CDK2 and mitogen-activated protein kinase p38 (P38) (22). Many of these inhibitors are being tested for the treatment of cancer including erlotinib (OSI-774, CP-358,774, Tarceva^TM), 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 (47, 48) and HER3 (49) 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 (25, 26), 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Purification of EGFRK-- DNA encoding residues 672-998 was amplified from full-length EGFR cDNA (27) 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 an XbaI site. The product was digested with NotI and XbaI 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 Five^TM 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 mM DTT. 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 mM sodium 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.

Structure Determination-- 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, 0.1 M 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 (Table I). Data were reduced with HKL2000 and Denzo/Scalepack (28). 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/(I)  2) rather than on agreement factors.

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) (29) 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 R_free, and the same set was used (and extended to higher resolution) for the synchrotron data sets. Initial phases treated with SIGMAA (30) 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 (31), 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 Ų), 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 (50) (PDB accession codes 1M14 and 1M17, respectively).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 -strands and one -helix (C), whereas the larger COOH-terminal lobe (C-lobe) is mostly -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).

View larger version (53K): [in this window] [in a new window]   Fig. 1.   The EGFRK structure with key features indicated. Erlotinib is found in the cleft between the amino-terminal and carboxyl-terminal lobes. The COOH-terminal ordered region including residues Leu977-Pro995 is not included here.

N-lobe-- The NH₂-terminal lobe of EGFRK adopts a tertiary structure similar to previously observed structures of RTKs (r.m.s. deviations for superpositioning C- 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 Ser⁷⁴⁴ also contribute. At Glu⁶⁸⁵, the polypeptide chain assumes a trace more similar to those of the lymphocyte tyrosine kinase (LCK, PDB code 3lck) (32), the insulin receptor kinase-phosphorylated form (33) (p-IRK, PDB code 1ir3), and the unphosphorylated form of the FGF receptor kinase (34) (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 -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 -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 - and -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 Lys⁷²¹ and 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.

C-lobe-- The COOH-terminal domain of EGFRK contains the usual organization of -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.

Activation Loop-- 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 (35). 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.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Activation loops. The close structural correspondence between the EGFRK A-loop (blue) and the A-loop from the phosphorylated form of the insulin receptor kinase (33) (gold) is shown. The hydrophobic interaction between Lys836 and Tyr845 almost exactly reprises that between Arg1155 and Tyr1163 of p-IRK (underlined). The presence of four glutamate residues in this part of EGFRK has been suggested as a cause for its intrinsic catalytic activity.

View larger version (72K): [in this window] [in a new window]   Fig. 3.   Representative electron density from the EGFRK/erlotinib structure. Map (2Fo  Fc 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).

Anti-parallel -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 -electrons of the tyrosine. Tyr⁸⁶⁷ -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 (36) (PDB code 1phk), casein kinase 1 (37) (PDB code 1csn), carboxyl-terminal Src kinase (38) (PDB code 1k9a), and twitchin kinase (39) (PDB code 1koa). The most relevant of these is the phosphorylase kinase in which a Glu residue (Glu¹⁸²) at an A-loop position analogous to sites of phosphorylation interacts with the Arg preceding the catalytic Asp and thus reprises the p-Tyr or p-Thr role. The EGFRK A-loop is rich in Glu residues, and the contribution of two of these residues (Glu⁸⁴² and Glu⁸⁴⁴) to EGFRK function was demonstrated using amino acid substitutions that altered the in vitro kinetics of phospho-transfer (40). In EGFRK, the structural roles of Tyr⁸⁴⁵ and Glu⁸⁴⁸ are a hybrid between the hydrophobic contacts of Tyr¹¹⁶³ in p-IRK and the electrostatic effects of Glu¹⁸² in phosphorylase kinase.

Overall, the conformation adopted by the EGFRK A-loop appears to result more from an energetic advantage for the active conformation rather than an energetic disadvantage for an alternate "inactive" one. For instance, there is no apparent impediment to the EGFRK A-loop adopting the conformation found for FGFRK from which it differs by up to 25 Å. Additionally, intermolecular crystal packing contacts do not seem to play a role, because influential interactions of this type do not exist in our structures and deleterious ones would not arise if we were to imagine an FGFRK-like A-loop in our crystal lattice.

LVI Motif-- The C-lobe of EGFRK also contains the distinctive three-amino acid sequence Leu⁹⁵⁵-Val⁹⁵⁶-Ile⁹⁵⁷ found previously to regulate in a poorly understood manner the transphosphorylation of substrate tyrosines in oligomerized EGFR family complexes. Of the three amino acids, substitutions for Leu⁹⁵⁵ are the most deleterious for phospho-transfer activity. A model suggesting direct contact between this segment and another protein, either another receptor or an unidentified adaptor protein, has been proposed for the ErbB2/ErbB3 system (13). In our EGFRK structures, LVI is in close contact with the C-lobe. The proximal polypeptide region is coupled to the C-lobe by the completely buried Leu⁹⁵⁵ side chain (Fig. 4). This strongly suggests that the Leu⁹⁵⁵ side chain does not contact another protein. It is more probable that the replacement of Leu⁹⁵⁵ with alanine uncouples it and nearby residues from the C-lobe. It is possible that the tripeptide segment mediates subtle allosteric effects in the intracellular region that affect the viability of the COOH-terminal tail as a substrate for transphosphorylation.

View larger version (99K): [in this window] [in a new window]   Fig. 4.   The LVI tripeptide segment of EGFRK is found in close association with the C-lobe. A solvent-accessible surface from EGFRK with LVI removed is depicted. Residue Leu955, the most important as gauged by mutagenesis studies, is found within what in its absence would be a hydrophobic pit.

Following the LVI sequence, the electron density continues strongly until dropping off abruptly after residue Asp⁹⁶⁰, and 13 residues following His⁹⁶⁴ have largely untraceable density. The disordered residues are part of an endocytotic signal sequence that directs ligand-activated receptors to recycling or degradation via clathrin-coated pits (41, 42). The 19 residues starting at Leu⁹⁷⁷ become increasingly well ordered and are in close contact with neighboring molecules (crystal packing contacts) beginning with Asp⁹⁸⁵. The last two amino acids in our construct are not observed. There is no discernable secondary structure in the COOH-terminal extension with the exception for a few isolated (i, i+4) -helical H-bonds. The crystal packing contacts experienced by the 19 well resolved COOH-terminal residues involve two neighboring molecules (Fig. 5). The first contact reduces solvent-accessible surface area by approximately 1300 Å² on each side, whereas the other is characterized by an ~1000-Ų reduction. The first contact, in addition to being of greater size, is also slightly more complimentary with respect to electrostatic charge. The COOH-terminal ordered region is highly acidic (LMDEEDMDDVVDADEYLIPQ), and the opposing contact region of the neighboring molecules is generally basic. The opposing region of the larger interface is at the back side of the ATP-binding cleft. The smaller contact area for the COOH-terminal ordered region is in the C-lobe of a different neighboring molecule.

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Stereoview of the intermolecular contacts of the extreme COOH terminus of the crystallized construct showing solvent-accessible surfaces and worm representations of the protein backbones. The reference molecule (blue) is shown from Leu977 to Pro995. There are 13 residues preceding Leu977 and two residues following Pro995 that were too poorly ordered to be fit. The yellow molecule shares a contact area of ~1300 Å2 (each side) with the blue one with specific contacts among Asp985/Lys715' (main chain/main chain), Val987/Lys715' (main chain/main chain), Asp988/Gln767' (side chain/side chain), Asp990/(Lys822' + Lys828') (side chain/side chain), and Tyr992/Glu712' (side chain/side chain). The green molecule shares ~1000 Å2 (each side) with the blue one, and contacts include Asp985/Lys946" (side chain/side chain), Glu991/Lys799" (side chain/side chain), and Leu993/Arg938" (main chain/side chain). The charge complimentarity for these interactions is good with a net-negative charge on the blue molecule and net-positive charges for contact regions from both neighboring molecules. Because of the disordered 13 amino acids preceding Leu977, the assignment of "reference" and neighbor is arbitrary with the result that the blue depicted here may also be considered to belong to either the yellow or the green molecules.

Catalytic Site-- The well conserved sequence of the EGFRK catalytic-loop (HRDLAARN), also present in p-IRK and FGFRK, shares structural conservation with these RTKs as well with a r.m.s. deviation of most atoms of only 0.1 Å. The nearby conserved DFG sequence, important for ATP coordination, adopts the p-IRK-like arrangement. The ATP binding site of apo-EGFRK would require only limited rearrangement to accommodate the AMP-PNP present in the p-IRK structure. The principle difference between the p-IRK and EGFRK structures in this region is in the nucleotide phosphate-binding loop. Although in both apo-EGFRK and EGFRK/erlotinib the nucleotide phosphate-binding loop is poorly ordered, Phe⁶⁹⁹ is better discerned in the erlotinib complex as it is brought closer to the C-lobe as a general consequence of inhibitor binding.

Relationship between N-lobe and C-lobe-- The opening angle between the two lobes of kinase structures has been observed to differ depending on the presence or absence of ATP or a close analogue. Those forms with smaller opening angles ("closed") bring the important catalytic elements into proximity. Among available prior kinase structures, the relationship between EGFRK lobes is most similar to those of LCK, p-IRK, and FGFRK for which superposition of ~250 C- pairs yields r.m.s. deviation values of ~1.2 Å. After superposition on the C-lobes, the -strands of both apo-EGFRK and EGFRK/erlotinib N-lobes align more nearly with those of LCK and p-IRK than those of FGFRK, and the placement of the C-helix is intermediate between those of LCK and p-IRK.

Comparing apo-EGFRK and erlotinib complex structures, the r.m.s. deviation for C- atoms excluding termini is 0.4 Å, and the r.m.s. deviations for the isolated lobes are 0.25 Å (C-lobe) and 0.5 Å (N-lobe). Most localized differences between the apo-EGFRK and erlotinib complex structures arise coincident with elevated B-factors and can be discounted. One exception is seen at Arg⁷⁵² where C- atoms are 1.5-Å apart. The validity of this interpretation of the respective electron density maps was gauged by inspecting an (F_o  F_o) difference map calculated using phases from the erlotinib complex. This map supports the variation at Arg⁷⁵² and more generally indicates a shift of the N-lobe that reduces the interlobe angle in EGFRK/erlotinib versus apo-EGFRK. This effect is small, and distances between corresponding C- atoms are ~0.6 Å on average, closing the interlobe angle by ~3°.

Inhibitor Interactions-- Previous studies have indicated that 4-anilinoquinazolines such as erlotinib cause inhibition through binding to the site occupied by ATP during phospho-transfer. There is a vast literature describing ATP-competitive inhibitors directed against the EGFRK (25, 26, 43, 44). Computational methods have been applied to quinazolines binding to EGFRK (25, 26), and where the results have been depicted, they differ in detail from our findings. Whether the differences result from inaccuracies in the protein homology model, the nature of quinazoline substituents or other causes is not clear. In our complex of erlotinib with EGFRK, we find the compound in an orientation very reminiscent of those seen for closely related 4-anilino-quinazoline molecules complexed with cyclin-dependent kinase 2 (CDK2) and mitogen-activated protein kinase p38 (P38) (PDB codes 1di8 and 1di9, respectively) (22). Erlotinib lies with the N1- and C8-containing edge of the quinazoline directed toward the peptide segment connecting N- and C-lobes, with the ether linkages projecting past the connecting segment into solvent and the anilino substituent on the opposite end sequestered in a hydrophobic pocket (Fig. 6). The N1 of the quinazoline accepts an H-bond from the Met⁷⁶⁹ amide nitrogen. The other quinazoline nitrogen atom (N3) is not within H-bonding distance of the Thr⁷⁶⁶ side chain (4.1 Å), but a water molecule bridges this gap. Such a water molecule was observed by Shewchuk et al. (22) in the P38-inhibitor complex and was predicted by Wissner et al. (26). The same water molecule contacts the side chain of Cys⁷⁵¹, which itself is disordered between two side chain conformers. The less robust nature of this water-mediated H-bond between erlotinib and EGFRK parallels the relatively small effect on inhibitor affinity seen for substitution with carbon for N3 among compounds characterized by Rewcastle et al. (45).

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Stereoview of the inhibitor binding site and nearby residues from EGFRK/erlotinib. Dashed line indicates an H-bond from the Met769 amide nitrogen to erlotinib. The light blue sphere is a water molecule. The electron density for the side chains of Cys751 and Asp831 was modeled using two conformers, but only one is depicted for clarity.

The interplanar angle of aromatic ring systems in erlotinib is 42°. This directs the acetylene moiety into a pocket that many kinase domains share when the amino acid side chain at position 766 is small (threonine in EGFRK). P38 also has threonine at this position, but CDK2 has a phenylalanine so that where the P38-bound inhibitor has a dihedral angle similar to ours (39°), in the CDK2-bound inhibitor the anilino ring is coplanar with its quinazoline. We find Thr⁷⁶⁶, Lys⁷²¹, and Leu⁷⁶⁴ are <4 Å from the acetylene moiety on the anilino ring (Thr⁷⁶⁶ and Leu⁷⁶⁴ are ~3.4 Å). Both Met⁷⁴² and Cys⁷⁵¹ have been suggested to contact inhibitors very similar to erlotinib (25), but contact distances seen here for both are greater than 4.5 Å. In the CDK2-inhibitor complex, quinazoline carbon atoms C2 and C8 direct their attached hydrogen atoms toward protein carbonyl oxygen atoms analogous to those of Gln⁷⁶⁷ and Met⁷⁶⁹, respectively, with carbon-oxygen distances of ~3 Å. In the P38 complex, only the first of these contacts is observed. In our complex, we observe both C2- and C8-carbonyl oxygen contacts (3.1 and 3.2 Å). Thus, the disposition of erlotinib with respect to the interplanar angle and linker-region contacts is a hybrid of the CDK2 and P38 quinazoline complexes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The inherent catalytic activity of the EGFR family kinases is unique among RTKs. The regulation of the vital cellular processes influenced by EGFR signaling must be exerted by control of the delivery of COOH-terminal substrate tyrosines to the active site. The structure we have determined makes this emphatically clear, as we find all of the catalytic elements primed and ready for phospho-transfer, even though crystals were grown without cofactor or substrate analogues. This fundamental difference from other RTKs raises the possibility that the spatial relationships between the two or more kinase domains in an EGFR family complex may differ from those of even closely related RTKs like FGFR, for instance.

The reduction in the interlobe angle upon the addition of erlotinib to apo-EGFRK crystals suggests that ATP binding would have a similar effect. Learning the true magnitude of such effects will require crystals grown with the ATP-binding cleft already occupied; however, no essential element of the catalytic machinery requires much change from the arrangements we see in this work. Crystals of the erlotinib complex were obtained by crystallizing EGFRK in the absence of inhibitor and then soaking in the inhibitor. Therefore, any artifact from the crystal lattice is more likely to have limited the observed interlobe shift than to have augmented it. "Closed" forms of protein kinase domains are found when both A-loop phosphorylation and ATP (or analogue) are present. The constitutive catalytic competence of EGFRK and our finding of the apo-EGFRK A-loop in an active conformation are consistent. Both the crystal lattice and the significant differences between erlotinib and ATP are likely to have limited the magnitude of the interlobe shift we observe upon inhibitor binding and suggest that our erlotinib complex may not represent the fully closed form but instead an intermediate of the transition.

Our finding that Leu⁹⁵⁵ is concealed in the COOH-terminal lobe argues against a direct binding role with either the partnered receptors or accessory proteins. A more plausible explanation for the impacts on transphosphorylation caused by substitution for Leu⁹⁵⁵ is an allosteric effect caused by the loss of the Leu⁹⁵⁵-C-lobe connection. It seems somewhat more plausible that such allosteric effects would arise in the COOH-terminal tail following Leu⁹⁵⁵. The intermolecular contact regions present in our crystal lattice are larger than is typical in protein structures. They are clearly required to form these crystals. The relevance of these contacts involving the COOH-terminal ordered region is difficult to gauge. The lack of traceable density for the preceding 13 residues presents the possibility that our arbitrary assignment of "self" and "neighbor" proteins is incorrect. The larger of the two contacts of the COOH-terminal ordered region is at a site on the neighbor protein that includes parts from both N- and C-lobes and the connecting segment between them, and thus, large changes in the interlobe relationship may disrupt this interaction. Such a scenario is consistent with previous results demonstrating in vitro dimer disruption upon ATP binding (46).

The present data do not permit us to conclude that these contacts also arise in a cellular context, but the high local concentration of membrane-bound EGFR and evidence for non-ligand induced dimers (4) suggest that close association among some parts of the intracellular domains is probable.

	ACKNOWLEDGEMENTS

We thank M. Ultsch, S. Hymowitz, and M. Franklin for data collection help, I. Massova for help with analysis as well as F. Rotella and the staff at Structural Biology Center, and T. Earnest, K. Henderson, and staff at Advanced Light Source. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Biological and Environmental Research under Contract No. W-31-109-ENG-38. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, the U. S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1M14 and 1M17) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ To whom correspondence should be addressed: 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-2106; Fax: 650-225-3734; E-mail: eigenbrot.c@gene.com.

Published, JBC Papers in Press, August 23, 2002, DOI 10.1074/jbc.M207135200

	ABBREVIATIONS

The abbreviations used are: 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'-(,-imino)triphosphate; CDK2, cyclin-dependent kinase 2.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R.-H. Tao and I. N. Maruyama All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells J. Cell Sci., October 1, 2008; 121(19): 3207 - 3217. [Abstract] [Full Text] [PDF]

				T. Trowe, S. Boukouvala, K. Calkins, R. E. Cutler Jr., R. Fong, R. Funke, S. B. Gendreau, Y. D. Kim, N. Miller, J. R. Woolfrey, et al. EXEL-7647 Inhibits Mutant Forms of ErbB2 Associated with Lapatinib Resistance and Neoplastic Transformation Clin. Cancer Res., April 15, 2008; 14(8): 2465 - 2475. [Abstract] [Full Text] [PDF]

				A. Kumar, E. T. Petri, B. Halmos, and T. J. Boggon Structure and Clinical Relevance of the Epidermal Growth Factor Receptor in Human Cancer J. Clin. Oncol., April 1, 2008; 26(10): 1742 - 1751. [Abstract] [Full Text] [PDF]

				E. R. Wood, L. M. Shewchuk, B. Ellis, P. Brignola, R. L. Brashear, T. R. Caferro, S. H. Dickerson, H. D. Dickson, K. H. Donaldson, M. Gaul, et al. 6-Ethynylthieno[3,2-d]- and 6-ethynylthieno[2,3-d]pyrimidin-4-anilines as tunable covalent modifiers of ErbB kinases PNAS, February 26, 2008; 105(8): 2773 - 2778. [Abstract] [Full Text] [PDF]

				C.-H. Yun, K. E. Mengwasser, A. V. Toms, M. S. Woo, H. Greulich, K.-K. Wong, M. Meyerson, and M. J. Eck The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP PNAS, February 12, 2008; 105(6): 2070 - 2075. [Abstract] [Full Text] [PDF]