The Juxtamembrane Segment Activates the Kinase Domain in Solution
The core kinase domain has low activity, when measured in solution using purified protein (the catalytic efficiency, kcat
, is 0.0049 ± 0.0005 s−1
, ). Introduction of the L834R mutation, commonly found in lung cancer patients, increases the activity of the kinase core by ~14-fold, consistent with previous results (Yun et al., 2007
; Zhang et al., 2006
) (). Attachment of the juxtamembrane segment to the wild type kinase domain results in substantially greater activity. The value of kcat
for the JM-kinase construct (0.33 ± 0.02 s−1
) is ~70-fold greater than for the kinase core alone. The activity of the kinase core is increased ~20-fold by concentrating it on lipid vesicles (Zhang et al., 2006
), but the addition of the juxtamembrane segment results in greater catalytic efficiency in solution, without concentration on vesicles ().
The Effect of the Juxtamembrane Segment on Activity
Dimerization and Activation Requires Intact Juxtamembrane Segments on both the Receiver and the Activator Kinase Domains
Deletion of the JM-A segment reduces catalytic efficiency by ten-fold, to 0.032 ± 0.003 s−1
(), showing that an intact juxtamembrane segment is required for full activation. In order to determine if the JM-A segment is required on both the activator and receiver, we made mutant kinases that can only take the activator or the receiver position. We refer to kinases with an I682Q mutation, which can take only the activator position, as “receiver-impaired” (Zhang et al., 2006
). Kinases with a V924R mutation cannot be activated, and are referred to as “activator-impaired”. Both the activator-impaired and receiver-impaired JM-kinase constructs have low activity alone, but mixing these constructs recovers ~50% of the activity of wild type JM-kinase constructs in solution, as observed in cell transfection studies (Thiel and Carpenter, 2007
) (). In contrast, activity is reduced significantly if the JM-A segment on either the activator-impaired or the receiver-impaired kinase is missing (). Thus, both JM-A and JM-B segments are required on both the activator and the receiver kinases for robust stimulation of activity.
The core kinase domain is a monomer, based on static light scattering at a concentration of 150 µM, whereas the JM-kinase construct is predominantly a dimer with no detectable monomer (Figure S1
). The specific activity of the JM-kinase construct increases with concentration, and the dissociation constant (KD
) for dimerization is estimated to be ~200 nM (). Deletion of the JM-A segment increases the midpoint value of the concentration dependence curve to at least ~8 µM (). The >40 fold increase in the estimated values for KD
for dimerization upon deletion of the JM-A segment provides strong evidence for a role of the JM-A segment in dimerization.
Activation by the Transmembrane and Juxtamembrane Segments Is Comparable to the Effects of Enforced Dimerization
We used cell-based assays to compare the activities of various constructs containing the JM-kinase portion of the receptor. One construct includes the transmembrane and intracellular domains of EGFR (TM-ICD). The other construct is a fusion of the intracellular domains to 29 residues of the coiled-coil portion of the transcriptional activator GCN4 (O'Shea et al., 1991
), which is expected to enforce constitutive dimerization on the intracellular domains.
We transfected COS7 cells with a ~5-fold lower level of DNA than used in the previous study (Thiel and Carpenter, 2007
) (). Under these conditions, the intracellular domains alone display very low levels of autophosphorylation in an immunoprecipitation assay. In contrast, a construct that contains the transmembrane segment, but without the extracellular segment (TM-ICD), shows high activity. The activity of the TM-ICD construct is about the same as that for the GCN4-ICD construct, indicating that the effect of the juxtamembrane segment, when fused to the transmembrane domain and localized to membranes, is comparable to that of enforced dimerization. This result is consistent with previous experiments showing that removal of the extracellular domains activates the kinase domains of EGFR (Chantry, 1995
; Nishikawa et al., 1994
; Zhu et al., 2003
Many kinases are activated by imposed dimerization, where the effect could simply be due to enhancement of trans-phosphorylation. Introduction of the V924R mutation in the GCN4-ICD construct, which is expected to disrupt the asymmetric dimer interface, results in a complete loss of activity (). Thus, even when dimerization is enforced by GCN4, formation of the asymmetric dimer appears to be essential for activity.
A Structure of the Her4 Kinase Domain Suggests a Latching Function for the JM-B Segment of the Juxtamembrane Domain
A crystal structure of the Her4 kinase domain bound to a covalently linked inhibitor (PDB ID: 2R4B) (Wood et al., 2008
) provides insight into the structural basis for the role of the juxtamembrane segment. Crystallization of the Her4–inhibitor complex inhibitor utilized a construct that includes the JM-B portion of the juxtamembrane segment (Wood et al., 2008
). Although the authors do not comment on this fact, the kinase domains in their crystal structure form an asymmetric dimer that is similar in general terms to that seen in other crystal structures for active forms of EGFR and Her4. The kinase domain of Her4 in this complex is in an inactive conformation, which is required for accommodation of the covalently bound inhibitor (Wood et al., 2008
). Nevertheless, the crystal lattice generates a “daisy chain” of kinase domains in which each molecule in the chain is docked on the next one, as in crystals of the active kinase domain.
The last ten residues of JM-B (corresponding to Gly 672 to Ile 682 in EGFR) have been visualized previously at the asymmetric dimer interface, and are critical for EGFR activation (Zhang et al., 2006
). What is new in the Her4-inhibitor complex is that the rest of the JM-B segment, provided by the kinase domain in the receiver position, latches two kinase domains together by running along the surface of the C-lobe of the activator kinase domain ().
Role of the Juxtamembrane Latch in Activation of EGFR
Formation of the juxtamembrane latch involves residues in the receiver and activator kinases that are conserved in EGFR family members (). The interaction involves several hydrogen bonds and hydrophobic contacts (see ). Mutations in C-lobe residues that anchor the JM-B region (e.g., Asn 972, Arg 949, Asp 950, Arg 953 in the C-lobe) have substantial inhibitory effects on EGFR autophosphorylation in cell-based assays (). Mutation of Glu 666 in the JM-B region, which forms an ion pair with Arg 949, is also inhibitory. Three hydrophobic residues in the JM-B segment (Leu 664, Val 656, Leu 668) are also essential for EGFR activation (). Leu 668 packs against Pro 951 in the C-lobe of the kinase domain. Leu 664 and Val 665 are located near the junction of JM-B with the N-terminal JM-A segment.
A segment spanning residues 315–374 of the EGFR inhibitor Mig6 blocks asymmetric dimer formation (Zhang et al., 2007
). A six residue motif (EPLTPS) in the juxtamembrane latch is almost identical in sequence with six residues in Mig6 (residues 323–327, with sequence EPLSPS). This sequence motif is located six residues upstream of the first ordered residue in the crystal structure containing Mig6, and so was not seen in that complex. The EPLSPS motif of Mig6 can be docked onto the C-lobe of the kinase domain based on the corresponding motif in the juxtamembrane latch, while allowing the rest of Mig6 to block the interface between the activator and receiver kinase domains (). This indicates that part of the function of Mig6 is to prevent formation of the juxtamembrane latch.
The Juxtamembrane Latch Involves the C-lobe of the Activator Kinase Domain, and not that of the Receiver
A crucial assumption in our subsequent modeling is that the JM-kinase portion of the receptor forms a closed dimer, in which the two juxtamembrane segments interact with each other rather than repeating identical interactions in a daisy chain. We used cell transfection assays to confirm that the juxtamembrane latch is engaged only by the activator and not by the receiver in activated full length receptors.
When EGFR variants that are receiver-impaired or activator-impaired are co-expressed, robust EGF-dependent activity is observed (). We introduced a mutation in the C-lobe of the kinase domain (R953A) that disrupts the juxtamembrane latch (see ). If the mutation is introduced only in the receiver-impaired EGFR construct, which presumably serves only as an activator, activity is reduced in co-transfection experiments. There is essentially no effect on EGFR activity if the R953A mutation is introduced in the activator-impaired kinase domain. These results are consistent with the formation of a closed dimer and imply that the juxtamembrane segment of the activator may be free to interact with the receiver.
The JM-A Segment is Likely to be Helical
The JM-A segments have three elements that are conserved among EGFR family members (). The N-terminal region contains 5–7 basic residues, including several arginines. At least some of these residues are likely to interact with phosphate groups in the membrane bilayer (McLaughlin et al., 2005
). The central portion contains hydrophobic residues in an i
, i+3, i+4
pattern within an LRRLL motif in EGFR. The C-terminal region contains acidic residues.
A Helical Dimer in the JM-A Segment
, i+3, i+4
pattern of hydrophobic residues in JM-A suggests an α-helical structure. The structure of a micelle-bound peptide containing the juxtamembrane segment of EGFR has been determined by NMR (Choowongkomon et al., 2005
). The relevance of this structure, in which the JM-A segment is helical, is uncertain because one portion of this peptide is normally integrated into the folded structure of the kinase domain core as a β strand, but instead adopts an α-helical conformation in this micelle-bound peptide.
We analyzed an isolated 15 residue peptide spanning the JM-A segment by solution NMR. These data provide evidence for the transient formation of an α-helix spanning the length of the peptide, and also for a concentration-dependent interaction between peptides (see Figure S2 and Supplemental Data
). We used cell transfection experiments to probe the conformation of the JM-A segment in the full length receptor, taking advantage of the fact that glycine residues weaken an α-helix but alanine residues do not (Pace and Scholtz, 1998
). We examined the activity of full length EGFR constructs with arginine residues in the 655
motif in JM-A replaced by glycines. This led to a significant reduction in EGFR activity ( and Figure S3A
). To account for the effects of charge removal, as opposed to helix weakening, we compared the effects of the glycine substitutions to that of alanine substitutions. The effect of alanine substitutions is smaller than that of glycine substitutions, consistent with a helical conformation for the JM-A segment ( and Figure S3A
The results of co-transfection experiments using activator-impaired and receiver-impaired kinases show that replacement of Arg 656 and Arg 657 by glycines on either the activator or receiver kinase individually also results in a reduction of EGFR activity relative to alanine substitutions ( and Figure S3A
). These data indicate that a helical conformation for the JM-A segment is important for both the activator and the receiver.
A Potential Antiparallel Helical Dimer Interaction in the Juxtamembrane Segment
Mutation of the three leucine residues in the LRRLL motif (residues 655, 658 and 659) to either alanine or aspartic acid attenuates the activity of the receptor (Figure S3B
). This led us to consider models for helical dimers in which the JM-A segments are packed closely together with a hydrophobic interface, in either an antiparallel or a parallel arrangement. We based our models on the structures of coiled coils (Woolfson, 2005
), but because we are considering only a short helical segment the predicted intermolecular contacts are similar for tightly packed straight helices. For each choice of orientation, parallel or antiparallel, there are two choices of sequence register, corresponding to whether the first leucine or the fifth one is at the a
position of a heptad sequence motif (Woolfson, 2005
If the orientation were parallel, several basic residues would be brought close together at the N-terminal ends of the two helices, which is likely to be energetically unfavorable. Only one inter-molecular ion pair is predicted for the parallel arrangement, and it involves Arg 662 and a glutamate sidechain (Glu 661 in one register and Glu 663 in the other; Figure S4A
). The parallel arrangement puts two such ion pairs close together by symmetry, as shown in Figure S4A
. Mutation of Arg 662 to glutamate would therefore place four glutamate sidechains in close proximity in the parallel dimer (Figure S4A
) which should destabilize the dimer. Mutation of Arg 662 to glutamate has little or no effect on EGFR activity (Figures S3B and S4B
), arguing against a parallel arrangement.
A parallel helical dimer is also inconsistent with the formation of heterodimers between the JM-A segments of EGFR family members. A single amino acid deletion in the JM-A segment of Her4 () results in Glu 693 of Her4 being directly apposed to another glutamate residue in modeled parallel heterodimers involving either Her2 or EGFR, in both possible sequence registers (Figure S4C
). In addition, no favorable intermolecular ion pairs are formed in these parallel heterodimers.
In an antiparallel coiled coil, residues at the a
positions of the heptad repeat in one helix interact with the residues at the d
positions, respectively, in the other (Woolfson, 2005
). Additional inter-helical interactions can be made by the sidechains at the e
positions. The LxxLL motif in EGFR JM-A fits into this pattern if the first leucine is at either the a
position or the d
position, with the second two leucines at the d
positions or the g
positions, respectively. We favor placement of the first leucine at the d
position because it provides a role for both arginine residues in the LRRLL motif, consistent with the strong effects of mutating these residues ( and Figure S3B
). In this pairing, Leu 658 and Leu 659 at the g
positions in each helix form a V-shaped crevice into which the sidechain of Leu 655 at the d
position from the other helix is inserted ().
An antiparallel dimer leads naturally to inter-helical ion pairing between arginine or lysine sidechains at the N-terminal end of each helix with the acidic sidechains located at the C-terminal end of the partner helix, and so we favor this arrangement for the helical dimer over a parallel one ( and Figure S5
). The pattern of inter-helical ion pairs predicted in this way for EGFR is also consistent with the formation of relevant heterodimers pairs (Figure S6
Additional support for the antiparallel model is provided by NMR measurements on a 35 residue peptide containing two copies of the JM-A segment of EGFR with a five residue flexible spacer (see Supplemental Data and Figure S7A
). The first and last leucine residues in the LRRLL motif in the first JM-A segment (Leu 655 and Leu 659) are labeled with 15
N. The second glutamate in the first segment (Glu 663) is labeled with 15
N and 13
C. As for the 15 residue JM-A peptide, NMR data for the tandemly linked JM-A segments provide evidence for transient rather than stable adoption of helical structure in both JM-A segments under the conditions of the NMR experiment (see Supplemental Data
). Despite the transient nature of these helices, the NMR data demonstrate that the two JM-A segments in the peptide interact with each other.
Data from Nuclear Overhauser Effect (NOE) experiments are consistent with an antiparallel rather than parallel orientation of two JM-A helices. This is not surprising, since an antiparallel interaction could occur in an intramolecular fashion within this peptide, whereas a parallel interaction would require dimerization. Nevertheless, the significance of these NMR data arises from the evidence that it provides for a specific register in the antiparallel interaction, in which the first leucine of the LRRLL motif is at the d
position of a heptad repeat, with the second two leucines at the g
positions, respectively ( and Supplemental Data
A Model for the Juxtamembrane Segment in the Context of the Asymmetric Kinase Domain Dimer
We joined the last residue in the modeled JM-A α-helix of the receiver kinase (the JM-A/receiver helix) to the first residue of the JM-B segment of the receiver kinase (the JM-B/receiver segment), while preserving the JM-B/receiver segment as seen in the Her4 structure (Wood et al., 2008
) (). Although the precise orientation of the JM-A/receiver helix with respect to the kinase domain is uncertain, we chose to orient this helix such that the sidechain of Glu 663 in the receiver helix points towards the sidechain of Lys 799 in the activator kinase domain, a residue that is crucial for activity (Zhang et al., 2006
). Another attractive feature of this orientation is that the face of the dimer that is likely to pack against the phospholipid bilayer has several arginine side chains, which could interact favorably with the membrane (). Mutation of many of these residues (e.g. Arg 651, Arg 657) results in greater reduction in EGFR activity than mutation of glutamate residues in JM-A, indicating that they are important for other functions in addition to dimerization. Particularly interesting is the presence of Thr 654 on this face of the dimer. Phosphorylation of Thr 654 attenuates EGFR signaling (Hunter et al., 1984
), and the model suggests that the addition of a phosphate group to this side of the helix dimer might weaken interaction with membranes, as shown for other membrane-interacting peptide segments (Thelen et al., 1991
Structural Coupling Between the Extracellular and Intracellular Domains in Active EGFR
Given the orientation of the JM-A/receiver helix, the antiparallel geometry naturally places the JM-A/activator helix between it and the first residue of the activator kinase domain core. In order to connect the activator kinase core to the JM-A/activator helix we modeled a 12 residue loop that connects Gln 677 of the activator kinase to Val 665 the JM-A/activator helix. We do not attach any particular significance to our modeled loop, except to note that the connection does not appear to impose unreasonable stereochemical constraints. We verified that the model we have constructed is without unnatural energetic strain by generating multiple molecular dynamics trajectories to relax the structure and noting that the dimeric structure is stable (see Supplemental Data and Figure S8
Potential Coupling Between the Juxtamembrane and Transmembrane Segments
The NMR-derived structure of the Her2 transmembrane helices (Bocharov et al., 2008
) shows that the transmembrane helices dimerize through a conserved glycine-containing motif (Burke et al., 1997
; Fleishman et al., 2002
; Sternberg and Gullick, 1989
). Alignment of the EGFR sequence onto the Her-2 NMR structure shows that the Cα atoms of the residues corresponding to Arg 647 in EGFR are ~20 Å apart. The distance between the Cα atoms of the N-terminal residues in our model for the two antiparallel JM-A helices (Arg 651) is 18 Å, and the three residues that bridge the gap between the juxtamembrane and transmembrane segments can do so readily (). The convergence between the transmembrane helical dimer and the ends of our modeled JM-A antiparallel dimer suggests that distortions in the relative orientations of the transmembrane helices could weaken the coupling to the juxtamembrane segments. Such misalignment might explain the position-sensitive effects of crosslinking or mutation in the transmembrane segments on EGFR activity (Bell et al., 2000
; Burke and Stern, 1998
; Moriki et al., 2001
Crystal structures of the extracellular domains of EGFR show that the active dimer brings the two C-terminal ends of the extracellular domains into close proximity (Burgess et al., 2003
). We propose that this conformation brings the transmembrane helices close enough together to dimerize via their N-termini, thus supporting the active juxtamembrane and kinase domain dimers. Our model therefore specifies how the activating signal is transmitted across the membrane ().
Crystal Structure of an Inactive Kinase Domain In a Dimer Form
We have obtained a crystal structure, at 3Å resolution, of an EGFR kinase domain variant inactivated by the V924R mutation. The kinase domains in this structure form a symmetrical dimer ( and Table S1
). Crystal structures of inactive forms of the EGFR kinase domain that have been determined previously have the kinase domain in essentially the same conformation as in our structure, but do not show extensive contacts within symmetrical dimers (Wood et al., 2004
; Xu et al., 2008
; Zhang et al., 2006
). These structures were determined in the presence of salts that might disrupt the electrostatic interactions that are at the center of the dimer interface of our crystal form, which is obtained under low salt conditions (see Experimental Procedures). A symmetrical EGFR dimer described previously has the kinase domains in an active conformation (Landau et al., 2004
A Symmetric Inactive Dimer of the EGFR Kinase Domain
Dimer formation in this new crystal form is mediated principally by the C-terminal tail of the kinase core (). There are four independent molecules in the asymmetric unit, designated A, B, C and D, which form two nearly identical dimers (A:B and C:D). In one molecule (A), electron density for the C-terminal segment is visualized up to Asp 990. The residues between Ser 967 and Met 978 form an α-helix (the AP-2 helix, see below), which is followed by a five residue turn spanning Asp 979 to Met 983. The last seven residues in the structural model, Asp 984 to Asp 990, run along the surface of the C-lobe of the kinase domain in an extended conformation. In molecule D there is no electron density for the extended strand (residues 984 to 990), and this region is blocked by crystal contacts. Electron density for the extended strand is present but weak in molecules B and C. Subsequent discussion of the dimer interactions is focused on the A:B dimer. The portion of the C-terminal tail (residues 967 to 983) that is of interest here is mainly disordered in structures of the active conformation of the EGFR kinase domain (Stamos et al., 2002
) and is partially ordered but in a different conformation in the other inactive structures (Wood et al., 2004
; Zhang et al., 2007
The Inactive Kinase Domain Dimer May Suppress Activity Prior to EGF Binding
Several studies have shown that EGFR dimerizes prior to EGF binding (Clayton et al., 2008
; Sako et al., 2000
). Although these preformed dimers are likely to involve the intracellular domain (Yu et al., 2002
), the orientation of the kinase domains in these preformed dimers is unknown. Our new inactive dimer has several features, which suggest it could play a role in the inhibition of kinase activity.
Each AP-2 helix in the C-terminal tail of one kinase subunit interacts with the other subunit, burying ~1400Å2
of surface area at each interface. The helix encompasses residues 973 to 977, which form the recognition element in EGFR for the AP-2 clathrin adapter protein (Sorkin et al., 1996
). The recognition of AP-2 by EGFR is dependent on activation by EGF (Sorkin and Carpenter, 1993
), and our structure shows how an inactive form of EGFR can sequester the AP-2 recognition motif. The interactions made by the AP-2 helix are reminiscent of those made by the SH2-kinase linker in inactive Src family kinases () (Sicheri et al., 1997
; Xu et al., 1997
). In particular, the engagement of the N-lobe of the adjacent kinase domain by the sidechain of Phe 973 in the C-terminal tail of EGFR is analogous to interactions made by the sidechain of Leu 255 in the SH2-kinase linker of c-Src (Xu et al., 1997
Acidic sidechains (Asp979, Glu 980, Glu 981) in the turn following the AP-2 helix form ion pairs with residues in the kinase domain (His 749, His 826, Lys 828 and Lys 822) (). The turn, referred to as an “electrostatic hook”, is located near the hinge region of the kinase domain and near the αC/β4 loop. In ZAP-70 and in certain other tyrosine kinases the formation of a hydrogen bonded network similar to that seen here in EGFR has been correlated with the inhibition of kinase activity (Chen et al., 2007
; Deindl et al., 2007
The conformation of the portion of the C-terminal tail that follows the electrostatic hook (residues 982 to 990) and runs along the surface of the C-lobe of the kinase mimics the manner in which the JM-B segment of the receiver kinase domain engages the same surface of the activator kinase domain when forming the juxtamembrane latch (). Thus, formation of the inactive dimer blocks formation of the activating juxtamembrane latch, in a manner similar to that postulated for Mig6 ().
Proposed Role of the Inactive Dimer in EGFR Autoinhibition
The key residues in the AP-2 helix, the electrostatic hook and the region of the C-terminal tail that interacts with the JM-B binding interface, as well as the basic residues that interact with the electrostatic hook, are conserved between EGFR and its kinase-active homologs (Her2 and Her4) (). The residues that form the electrostatic hook are absent in Her3, as are two of the three basic residues in the kinase domain. Presumably, the lack of kinase activity in Her3 renders inhibition of kinase activity unnecessary.
The surface electrostatic potential of the inactive dimer is strongly polarized ( and S9
). Eight lysine residues (residues 689, 692, 704, 715 in each subunit) are clustered together on the face of the dimer that is opposite to the internally engaged position of the C-terminal tail, which is a region of negative electrostatic potential. The lysine residues are conserved among EGFR family members. We speculate that the inactive dimer might be oriented with respect to the membrane such that the lysine residues can interact with negatively charged lipid head groups. The juxtamembrane segments would then be located on the far side of the dimer with respect to the membrane and dimerization of the JM-A segments would be prevented ().
Effect of Mutations in the C-terminal Tail on Activity of EGFR
Cell transfection experiments show that mutations in the electrostatic hook result in substantial activation of EGFR in the absence of EGF, as well an increased response to EGF, consistent with a role for this region in inhibiting EGFR kinase activity (). Notably, several insertions in exon 20 that drive constitutive EGFR activation are detected in lung cancer patients (Shigematsu et al., 2005
)((Greulich et al., 2005
). These insertions are in the β4/αC loop and are likely to disturb the electrostatic hook. Mutation of Lys 828, located within the electrostatic hook, increases the basal level of EGFR autophosphorylation (Zhang et al., 2006
Surprisingly, deletion of the AP-2 helix or mutations of residues within this helix resulted consistently in impaired EGFR function in the cell-based assay (Figure S10A and S10B
). These mutations result in activation in vitro
), demonstrating that the AP-2 helix is not required for the integrity of the catalytic domain. Mutation of residues in the AP-2 recognition motif does not lead to an obvious change in kinetics of EGFR internalization, presumably because of redundancy in endocytic targeting signals (Sorkin et al., 1996
). The observed reduction in activity in cell-based assays suggests that the AP-2 helix plays a role in the activation mechanism, perhaps by presenting the C-terminal tails for phosphorylation.