A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases
2006; Springer Nature; Volume: 25; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7601315
ISSN1460-2075
AutoresSilke Wiesner, Leanne E. Wybenga‐Groot, Neil Warner, Hong Lin, Tony Pawson, Julie D. Forman‐Kay, Frank Sicheri,
Tópico(s)Chromatography in Natural Products
ResumoArticle14 September 2006free access A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases Silke Wiesner Silke Wiesner Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Leanne E Wybenga-Groot Leanne E Wybenga-Groot Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Search for more papers by this author Neil Warner Neil Warner Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Hong Lin Hong Lin Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Tony Pawson Corresponding Author Tony Pawson Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Julie D Forman-Kay Corresponding Author Julie D Forman-Kay Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Frank Sicheri Corresponding Author Frank Sicheri Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Silke Wiesner Silke Wiesner Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Leanne E Wybenga-Groot Leanne E Wybenga-Groot Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Search for more papers by this author Neil Warner Neil Warner Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Hong Lin Hong Lin Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Search for more papers by this author Tony Pawson Corresponding Author Tony Pawson Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Julie D Forman-Kay Corresponding Author Julie D Forman-Kay Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Frank Sicheri Corresponding Author Frank Sicheri Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Silke Wiesner1,‡, Leanne E Wybenga-Groot2,‡, Neil Warner2,3, Hong Lin1, Tony Pawson 2,3, Julie D Forman-Kay 1,4 and Frank Sicheri 2,3 1Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada 2Program in Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada 3Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada 4Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada ‡These authors contributed equally to this work *Corresponding authors: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5. Tel.: +1 416 586 8471; Fax: +1 416 586 8869; E-mail: [email protected] for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Tel.: +1 416 813 5358; Fax: +1 416 813 5022; E-mail: [email protected] or Tel.: +1 416 586 8262; Fax: +1 416 586 8869; E-mail: [email protected] The EMBO Journal (2006)25:4686-4696https://doi.org/10.1038/sj.emboj.7601315 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix αC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix αC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation. Introduction Eph receptors regulate numerous developmental and cellular processes such as cell attraction/repulsion, adhesion/detachment and migration, thereby influencing morphogenesis and organogenesis (Kullander and Klein, 2002). The Eph receptor tyrosine kinase (RTK) family is divided into two groups, A and B, based on sequence similarity and a preference for binding to their ligands, the A- and B-type ephrins, respectively (Eph Nomenclature Committee, 1997). The domain structure of Eph RTKs is highly conserved from worm to human and consists of an extracellular N-terminal ligand-binding domain, a cysteine-rich region, two fibronectin type III repeats, a single membrane-spanning segment, a juxtamembrane segment (JMS), a tyrosine kinase domain (KD), a SAM domain and a C-terminal PDZ domain binding motif. Binding of ephrins to the extracellular domain of the Eph receptors leads to receptor multimerization and the activation of the cytoplasmic KD. Catalytic activation correlates with auto-phosphorylation on two tyrosines (Tyr604 and Tyr610, for murine EphB2) within the JMS (Ellis et al, 1996; Zisch et al, 1998; Binns et al, 2000) and possibly on a conserved residue, Tyr788, within the KD activation segment (referred to herein as Yact). In the absence of phosphorylation, the JMS represses the catalytic function of the adjacent KD through an intramolecular mechanism (Binns et al, 2000; Wybenga-Groot et al, 2001). The crystal structure of an unphosphorylated EphB2 fragment comprised of the JMS and the KD (EphB2 JMS-KD) revealed an auto-inhibitory state, in which an ordered α-helical JMS intimately associates with the KD (Wybenga-Groot et al, 2001). The majority of JMS contacts were directed at the N-lobe of the KD centered on helix αC and the adjacent β4-strand. Interestingly, helix αC possessed a kink not previously observed in active state protein kinase structures. In addition, more limited JMS contacts with the KD C-lobe appeared to prevent the activation segment from adopting an ordered productive conformation. Lastly, the JMS bridged the N- and C-lobes of the KD and thereby seemed to restrict inter-lobe flexibility. Together, these disruptive features were proposed to account for the repressed catalytic function of Eph receptors in their auto-inhibited states. Based on comparisons with the active insulin RTK structure (Hubbard, 1997) and structure-based mutational analyses, a two-component mechanism of catalytic activation was proposed for the Eph receptor family whereby phosphorylation of Tyr604 and Tyr610 within the JMS would cause the dissociation of the JMS from the KD (Wybenga-Groot et al, 2001). This event would relieve the distortion to helix αC and steric effects preventing the ordering of the activation segment. The latter, possibly supported by phosphorylation of Yact, would then allow the activation segment to adopt a productive conformation. However, a more recent crystal structure of a non-phosphorylated fragment of EphA2 containing exclusively the KD revealed a kinked helix αC and a significantly disordered activation segment (Nowakowski et al, 2002). Nevertheless, the EphA2 crystallization construct displayed robust catalytic activity in solution. These results have led us to experimentally revisit the mechanisms by which Eph RTKs are regulated. Here, we extend our analysis of the Eph receptor catalytic switching mechanism through a combination of mutational analyses, crystal structures and NMR studies of multiple EphB2 and EphA4 receptor proteins corresponding to auto-inhibited and active states. Using NMR spectroscopy, we provide direct evidence that phosphorylation of the JMS residues Tyr604 and Tyr610 induces disorder of the JMS and its dissociation from the KD. Our X-ray analyses show that JMS dissociation occurs without major structural changes to helix αC in the KD, but with partial ordering of the activation segment. Furthermore, NMR studies provide conclusive evidence that kinase activation coincides with increased inter-lobe flexibility and that helix αC samples conformations in solution that deviate from the kinked conformation observed in the crystal structures. In light of these data, we propose a refined model for Eph receptor regulation that suggests new avenues for further research. Results Mutational analysis of Tyr750 In the catalytically repressed EphB2 crystal structure, the C-lobe residue Tyr750 lies in direct vicinity to the JMS at the inter-lobe cleft and physically prevents the activation segment from adopting a productive conformation (Wybenga-Groot et al, 2001). Furthermore, Tyr750 in EphB2 is detectably phosphorylated in vivo and, therefore, could serve a phosphoregulatory function (Kalo and Pasquale, 1999). To specifically investigate the functional importance of Tyr750 in Eph receptor regulation, we first mutated this residue to Phe in an EphA4 JMS-KD receptor fragment (Figure 1A). For clarity, we employ EphB2 numbering with the corresponding EphA4 numbering in parentheses (the JMS-KD of EphA4 and EphB2 share 84% sequence identity). If phosphorylation of Tyr750 (Tyr742) was required for full activation, it would be expected that a Tyr750Phe mutant would be less active than wild-type EphA4 JMS-KD, whereas a Tyr750Glu mutant (mimicking phosphorylation at this position) would restore activity to a JMS-KD protein that is constitutively repressed by an additional Tyr604/610Phe (Tyr596/602Phe) double mutation. The latter substitutions were previously shown to completely repress catalytic function both in vitro and in vivo by preventing auto-phosphorylation on the JMS regulatory sites (Wybenga-Groot et al, 2001). The two purified EphA4 mutant proteins were tested for their ability to auto-phosphorylate (Figure 1A), to phosphorylate enolase (data not shown) and to phosphorylate a peptide substrate in a continuous spectrophotometric assay (Figure 1B). The EphA4 Tyr604/610Phe double mutant and wild-type JMS-KD proteins were analyzed concomitantly as reference points for the repressed (non-activatable) and active (fully activatable) states, respectively. A schematic of all Eph receptor constructs used in this study is shown in Supplementary Figure S1. Figure 1.The Tyr750Ala mutation increases Eph RTK activity. (A) Purified EphA4 proteins were assessed for their ability to auto-phosphorylate in an in vitro kinase assay (top panel). Coomassie-stained SDS–PAGE analysis (lower panel) shows that equal quantities of EphA4 proteins were employed in each kinase reaction. Lanes labeled as in (B). (B) Histogram of the specific activities of purified EphA4 proteins measured by the spectrophotometric coupling assay at 0.5 mM S1 peptide and 0.5 μM EphA4 kinase protein. Velocities were normalized to the specific activity of wild-type EphA4 (top panel). (C) Full-length EphA4 wild-type and mutant receptors were expressed in COS-1 cells and immunoprecipitated. The immunoprecipitates were resolved by SDS–PAGE and immunoblotted with anti-EphA4 (top panel) or anti-phospho tyrosine (lower panel). (D) EphA4 immunoprecipitates were assessed for their ability to auto-phosphorylate and phosphorylate enolase by an in vitro kinase assay (top panel). Coomassie-stained SDS–PAGE analysis (lower panel) shows that equal quantities of enolase were employed in each kinase reaction. Download figure Download PowerPoint Mutation of Tyr750 to Phe in the wild-type JMS-KD background did not adversely affect EphA4 kinase activity (Figure 1A and B, lanes 1 and 5). This suggests that Tyr750 is not a phosphoregulatory site or that, minimally, phosphorylation of Tyr750 is not essential for the catalytic activation of EphA4 JMS-KD in vitro. Mutation of Tyr750 to Glu in the repressed JMS background (Tyr604/610Phe) did not robustly rescue catalytic function in either kinase assay (Figure 1A and B, lane 4) and the same mutation in the wild-type background did not increase catalytic function (Supplementary Figure S2). These results further suggest that Tyr750 does not serve an essential phosphoregulatory function. In contrast to the Tyr750Glu mutant, a Tyr750Ala mutation fully rescued catalytic function of the constitutively repressed Tyr604/610Phe mutant to wild-type levels (Figure 1B, lane 3). As two prominent auto-phosphorylation sites have been removed from this protein (Y604/610), the level of auto-phosphorylation was expected to decrease relative to its wild-type counterpart (Figure 1A, lane 3). Accordingly, the near absence of phospho-tyrosine incorporation into all Tyr604/610Phe mutants (Figure 1A, lanes 2, 3, 4, 6 and 7) suggests that sites other than Tyr604 and Tyr610 (most importantly, Yact in the activation segment) are not significant targets for auto-phosphorylation in vitro. Supporting this notion, we find that the EphA4 JMS-KD fragment efficiently phosphorylates short peptides encompassing the JMS tyrosines 604 and 610 but not peptides containing the conserved tyrosine 750 and tyrosine 788 (Yact) of EphA2, EphA4 and EphB2 receptors (Supplementary Figure S3). Overall, our results indicate that a bulky side chain at position 750 is required for the repression of catalytic function by an unphosphorylated JMS. To test whether these in vitro results are relevant to the full-length receptor in vivo, we introduced Tyr750 mutations into full-length EphA4 receptor and investigated the properties of these mutants by transient transfection in COS-1 cells (Figure 1C and D). Overexpression of wild-type Eph RTKs in such cells yields constitutive auto-phosphorylation on Y604/610 and, hence, receptor activation without a need for ephrin stimulation. EphA4 variants were immunoprecipitated and then analyzed for auto-phosphorylation activity and phosphorylation of enolase substrate (Figure 2C and D). Mutation of Tyr750 to either Ala or Phe in an otherwise wild-type receptor did not perturb catalytic function as compared to wild-type EphA4 (Figure 1C, lanes 1–3). Consistent with the in vitro results, mutation of Tyr750 to Ala in the fully repressed (Tyr604/610Phe) full-length EphA4 produced a highly active receptor, as indicated by the extent of enolase phosphorylation (Figure 1D, lane 5). As observed for the JMS-KD fragment, the auto-phosphorylation level for this mutant was virtually undetectable (Figure 1C, lane 5). Again, these observations suggest that the activation segment is not a significant auto-phosphorylation site in vitro or in vivo. In contrast, mutation of Tyr750 to Phe in the repressed (Tyr604/610Phe) JMS background did not rescue catalytic activity (Figure 1D, lane 6). Similar results were obtained for full-length variants of the EphB2 receptor transiently transfected into 293T cells (data not shown). These findings support a role for Tyr750 in either composing the binding site for the JMS and/or in physically blocking the ordering of the activation segment in response to the binding of the JMS to the KD. Crystallographic analyses of active forms of EphB2 and EphA4 Despite the absence of the JMS, the crystal structure of the isolated EphA2 KD displays great similarity to the auto-inhibited EphB2 JMS-KD structure (Nowakowski et al, 2002). In particular, helix αC remains kinked and much of the activation segment is disordered. To investigate whether the active EphA2 KD structure is representative of other Eph receptor active states, we crystallized the isolated KD of EphB2 (aa 622–906) and an EphA4 JMS-KD fragment, bearing the kinase-repressing Tyr604/610Phe double mutation, but activated by a Tyr750Ala mutation (described above) (Supplementary Figure S1), and determined their X-ray structures by molecular replacement (see Supplementary data for details). A summary of the data collection and refinement statistics is given in Table I. As expression of catalytically active variants of EphB2 (but not EphA4) is toxic to bacteria, we introduced a kinase inactivating Asp754Ala mutation far removed from the JMS binding site in all EphB2 crystallization and NMR constructs described herein (Supplementary Figure S1). This mutation of the putative catalytic base (Hanks and Hunter, 1995) is predicted to inactivate the kinase without perturbing the structure of the KD itself. Table 1. Data collection, structure determination and refinement statistics for active Eph receptor KD structures EphB2 D754A KD (ADP) EphA4 Y604/610F, Y750A JMS-KD Wavelength λ=1.54180 Å λ=1.54180 Å Space group P21 P21 Resolution (Å) 2.6 2.35 Reflections, total/unique 428 269/41 270 26 864/9232 Completeness (%)a 99.8 (99.5) 88.6 (54.9) Rsym (%)a,b, a,b 8.2 (24.9) 5.7 (12.8) 〈I/σ〉a 16.9 (4.3) 17.2 (4.5) Refinement Resolution range (Å) 24–2.6 30–2.35 Reflections All data 40 244 9085 ∣F∣>2σ 37 259 8467 Rfactor/Rfree (%)c All data 20.4/26.1 20.9/24.8 ∣F∣>2σ 19.4/25.2 20.0/23.9 Residues in disallowed/most favored regions of the Ramachandran plot 0/88.0% 0.9/86.8% Average B-value (Å2) 31.6 42.4 RMSD for main-chain B-values (Å2) 0.98 1.00 RMSD for side-chain B-values (Å2) 1.59 1.54 RMSD for bonds (Å) 0.008 0.006 RMSD for angles (deg) 1.09 0.96 Number of non-hydrogen protein atoms 8155 1968 Number of non-hydrogen nucleotide atoms 108 — Number of water molecules 301 (+4 Mg2+) 51 a Numbers given in parentheses refer to data for the highest resolution shell. b Rsym=100 × ∑∣I−〈I〉∣/∑〈I〉, where I is the observed intensity and 〈I〉 is the average intensity from multiple observations of symmetry-related reflections. c Rfree value was calculated with 10% of the data for EphB2 D754A KD, and 6% of the data for EphA4 Y604/610F, 750A JMS-KD. In brief, the refined EphB2 KD crystallographic model consists of four KD molecules in the asymmetric unit (referred to as molecules A–D), each with one ADP molecule and one magnesium ion bound. This model was refined to 2.6 Å to an Rfactor/Rfree 20.4%/26.1% (Table I). The four EphB2 KD structures are well ordered, except for five C-terminal residues and residues 774–796 corresponding to the kinase activation segment. The EphA4 Tyr604/610Phe, Tyr750Ala JMS-KD fragment, which contains one molecule in the asymmetric unit, was refined to 2.35 Å to an Rfactor/Rfree of 20.9/24.8% (Table I). Overall, the KD in the EphA4 structure is well ordered, with the exception of residues 651–654 (642–646) connecting β-strands 2 and 3, residues 665 and 666 (657–658) connecting strand β3 and helix αC, residues 779–794 (771–784) of the activation segment and eight C-terminal residues. Most notably, the JMS (599–621 in EphB2; 591–613) is almost completely disordered in this model, suggesting that the Tyr750Ala mutation restores catalytic function at least in part by directly perturbing the JMS–KD interaction. Overview of the active state Eph structures. The active state KD structures of EphA4, EphB2 and for comparison EphA2 (with two molecules in the asymmetric unit denoted A and B, PDB ID 1MQB) (Nowakowski et al, 2002) adopt canonical bi-lobal folds (Figure 2A). The N-lobe consists of a twisted five-strand anti-parallel β-sheet and a single helix αC, whereas the C-lobe is predominantly α-helical. The N- and C-lobes are connected by a short linker termed the hinge. Flexibility about the hinge is known to allow for a range of conformations, with the catalytically competent conformation typically corresponding to a closed conformation (Huse and Kuriyan, 2002). Comparison of the KDs in the EphA4 Y604/610F, Y750A JMS-KD and EphB2 KD structures reveals a greater degree of variability in lobe closure for the active state structures than displayed by the two unique molecules in the auto-inhibited EphB2 JMS-KD crystal structure (denoted A and B, PDB ID 1JPA) (Figure 2A and Supplementary Table SI). Although the degree of lobe closure for each active state structure is influenced in part by crystal packing interactions (as evidenced by differences between EphB2 KD molecules A–D) and the presence (EphB2) or absence (EphA4 and EphA2) of bound nucleotide, it is also conceivable that the greater range of lobe closures for the active state structures reflects an absence of the JMS, which by bridging both catalytic lobes would restrict inter-lobe flexibility. With the exception of the JMS and the activation segment, no gross conformational changes are observed in the N- or C-lobe of the KDs relative to the auto-inhibited EphB2 crystal structure (Figure 2A). However, the N-lobe appears to be slightly more affected than the C-lobe by the attainment of an active state as evidenced by a greater average RMSD between active and inactive state structures for the N-lobe (0.54 Å2) than for the C-lobe (0.33 Å2) (Supplementary Table SI). This is attributable to small differences in the relative position of helix αC with respect to the adjacent β-sheet of the N-lobe (Figure 2A). Figure 2.Comparison of Eph receptor KD crystal structures. (A) Superposition of active Eph KD structures with auto-inhibited EphB2 structures (PDB ID 1JPA). KDs were aligned using Cα atoms of the C-lobes (left panel) and Cα atoms of the N-lobes (right panel). Spheres represent the ordered boundaries of the KD activation segment. (B) Stereo view of kinked KD helices αC. The Eph receptor KDs (colored as in panel A) were superimposed using Cα atoms of helix αC. The kink stabilizing side chains of Ser677 and Ser680 in auto-inhibited EphB2 JMS-KD are shown in dark blue. (C) View of the inter-lobe cleft, highlighting the ordered regions of the KD activation segments (colored as in panel A). (D) Superposition of EphB2 JMS-KD with the active Eph KD structures, highlighting the region surrounding Tyr750. Backbone traces are colored as in panel A, with all side chains colored according to their respective backbones. The backbone of a typical activation segment conformation from the active insulin RTK (1IR3) is shown in magenta. Download figure Download PowerPoint An increase in inter-lobe flexibility for the active state EphB2 structures relative to the auto-inhibited EphB2 structures gives rise to a binding mode for the nucleotide that is more consistent with catalytic activity. In contrast to the auto-inhibited EphB2 structures (Figure 2), which were characterized by the presence of an ordered adenine base and complete disorder of the sugar and phosphate groups of the non-hydrolyzable ATP analogue AMP-PNP, the entire bound ADP molecule is ordered in all four EphB2 active state structures (Supplementary Figure S4). This change in nucleotide binding arises from an increase in lobe closure, which effectively translates the G-loop and invariant Lys–Glu salt bridge (between subdomains II and III) 2.4 Å (±0.3 Å) and 1 Å (±0.3 Å) toward the C-lobe. Although ATP binding appears more productive in the active state EphB2, two features differentiate the KD structure from prototypical active state conformations (such as that observed for PKA). Firstly, a kink in the N-lobe helix αC, previously attributed to the Eph receptor auto-inhibited state, persists in all seven active state structures (Figure 2B). In addition, the peptide substrate docking site, comprising the C-terminal portion of the activation segment, is disordered in all seven active state structures. A kinked helix αC and disordered activation segment are not likely catalytically competent conformations but may be dominant conformations (enthalpically favorable) in solution for the ‘active state,’ with catalysis occurring upon transient sampling of unkinked αC and ordered activation segments (see below). Role of Tyr750 in activation segment conformation. Disorder of the activation segment is a common feature of many inactive and far fewer active kinase state structures (Nolen et al, 2004). Figure 2C displays a superposition of the activation segment region of all active and auto-inhibited Eph receptor KD structures. Interestingly, although the majority of the activation segment is disordered for all structures, the extent of disorder is reduced in all the active state structures relative to auto-inhibited state structures. An increase in the order is observed for one to seven N-terminal and up to two C-terminal residues of the active state activation segments (Supplementary Table SI). The more extensive ordering of the activation segment of EphA2 molecule B correlates with a change in the Tyr750 side chain rotamer that allows for a productive path of the activation segment (Figure 2D). Importantly, the ability of Tyr750 to adopt a non-impeding rotamer is dependent on the dissociation of the JMS from the KD, as the Tyr750 side chain and the JMS would otherwise occupy the same physical space. In the only other active state structure with extensive ordering of the activation segment, the EphA4 Y604/610F, Y750A JMS-KD fragment, the bulky Tyr side chain of position 750 is entirely absent owing to its substitution by Ala. The correlation of Tyr750 position with more extensive ordering of the activation segment lends support to a role for Tyr750 in modulating activation segment conformation in response to JMS dissociation from the KD. In summary, our crystallographic analyses of Eph active state structures reveal evidence of an increase in inter-lobe flexibility, a partial ordering of the activation segment and the maintenance of a fully kinked helix αC. Subtle structural plasticity in the position of helix αC with respect to the N-lobe β-sheet structure is also apparent, which together may account for the enhanced catalytic function of our active state EphB2 protein constructs. NMR studies of inhibited and activated forms of EphB2 Eph receptor activation is hypothesized to involve a transition of the JMS from an ordered KD-associated state to a disordered state. We applied NMR spectroscopy to further investigate this issue, as changes in protein conformation and dynamics are readily identified by changes in the amide resonances in 1H,15N-correlation (HSQC) spectra. In order to localize regions involved in conformational change in an HSQC analysis (i.e. a chemical shift perturbation study), a full assignment of protein kinase amide resonances in at least one functional state (auto-inhibited or active) is required. To enable NMR studies of the Eph RTK system, we identified conditions under which an unphosphorylated and thus auto-inhibited EphB2 JMS-KD fragment could be expressed and refolded in sufficient quantities (see Supplementary data for details). This construct then served as a point of reference for the analysis of active state models described below. Characterization of the auto-inhibited EphB2 JMS-KD Fragment. A 2H,13C,15N-labeled protein sample of auto-inhibited EphB2 JMS-KD was prepared and more than 90% of all 1HN,13C,15N backbone resonances were assigned using relaxation-optimized (TROSY) heteronuclear NMR experiments. No resonance assignments could be obtained for the first five residues of the expression construct, residues 652, 666 and 724 in loop regions, the N-terminal half of the activation segment (aa 772–782) and three residues that follow proline residues (aa 798, 835 and 861). As the inability to assign many of these residues likely reflects conformational dynamics, it is worth noting that the N-terminal region of the JMS, the β2/β3-loop containing Gly652 and the entire activation segment were disordered in the auto-inhibited EphB2 crystal structure (Wybenga-Groot et al, 2001). Although we could assign the C-terminal half (aa 783–796) of the activation segment, reduced signal intensities, random coil 13Cα and 13Cβ chemical shifts (Supplementary Figure S5A) and 15N relaxation properties (unpublished results) indicate substantial disorder for these residues, which again is in agreement with the auto-inhibited EphB2 crystal structure. Overall, comparison of the assigned 13Cα and 13Cβ resonances with random coil values (secondary chemical shifts) (Supplementary Figure S5A) shows that the secondary structure of the auto-inhibited EphB2 JMS-KD in solution is very similar (if not identical)
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