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1 Supplementary Information to Wiesner et al.: A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases Supplementary Material and Methods Cloning and Mutagenesis Site-directed mutagenesis was performed using a PCR-based approach for all described mutations. All clones were confirmed by DNA sequencing. To generate a kinase-dead EphB2 KD fragment, first a D754A mutation was introduced into a previously described murine Y/F EphB2 -KD construct in a pgex-4t1 vector (Wybenga-Groot et al., 2001). The was then deleted giving rise to EphB2 D754A KD (residues ). A previously described murine EphA4 Y/F -KD construct in a pgex-4t2 vector (Wybenga- Groot et al., 2001) was used to introduce the following mutations (the murine EphB2 numbering scheme is used for consistency, with the corresponding EphA4 residue numbers listed in parentheses): (1) Y750A (Y742A), (2) Y750E (Y742E), (3) Y788F (Y act ; Y779F) and (4) Y750A/Y788F (Y742A/Y779F). As well, Tyr750 was mutated individually to Phe in the wild-type background. Full-length murine EphA4 cdna obtained from Regeneron (N. Gale) was subcloned into the mammalian expression vector pcdna3.1 (Invitrogen). The following mutations were generated in full-length EphA4: (1) Y750A, (2) Y750F, (3) Y/F, (4) Y/F, Y750A, and (5) Y/F, Y750F. For NMR studies, the Y/F EphB2 -KD fragment including the D754A mutation (aa ; EphB2 D754A -KD) was amplified by PCR and cloned into a pdest17 Gateway vector (Invitrogen). Phe/ were then mutated back to tyrosines using 1

2 the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The EphB2--KD Y750A mutant was obtained from the pdest17 Gateway EphB2 D754A -KD construct using the QuikChange Site-Directed Mutagenesis Kit. Using the EphB2 D754A -KD construct as PCR template the EphB2 KD fragment (residues ) was cloned into a pdest17 Gateway vector. Protein Purification for NMR studies Recombinant protein was purified by Ni-affinity chromatography from inclusion bodies solubilized in 20 mm sodium phosphate buffer ph 7.4, 0.5 M NaCl, 5 mm imidazole, 5 mm DTT and 6 M guanidinium hydrochloride. Denaturing purification also functioned to reintroduce exchangeable amide protons following expression in D 2 O. Purified EphB2 fragments were subsequently refolded at a concentration of ~0.5 mg/ml by dialysis at 4 ºC in (1) 50 mm Tris-HCl ph 8.0, 20 mm NaCl, 80 mm KCl, 5 mm DTT, 0.75 M guanidinium hydrochloride and 0.8 M arginine, (2) 50 mm Tris-HCl ph 8.0, 20 mm NaCl, 80 mm KCl, 5 mm DTT, and 0.4 M arginine, (3) 20 mm HEPES ph 7.2, 150 mm NaCl, 5 mm DTT. Refolding conditions were identified using the ProMatrix (Pierce) and FoldIt (Hampton Research) refolding screen following manufacturers instructions. After TEV cleavage EphB2 fragments were separated from the His-tag and His-tagged TEV protease by Ni-affinity chromatography followed by size-exclusion gel filtration. For preparative phosphorylation of EphB2 -KD, we employed a murine GSTtagged EphA4 kinase fragment (aa ) overexpressed in E. coli BL21(DE3) CodonPlus cells and purified as described (Binns et al., 2000). For the phosphorylation reaction, 10 µm EphB2 -KD was incubated with 2 µm GST-tagged EphA4 kinase in 50 mm HEPES (ph 7.2), 20 mm MgCl 2, 5 mm ATP, 5 mm DTT at 20 ºC for 2 hours. Complete phosphorylation 2

3 was verified by quantitative shift of EphB2 -KD on SDS-PAGE. GST-tagged EphA4 kinase was removed from the phosphorylated EphB2 -KD fragment using glutathione resin followed by size-exclusion gel filtration. 2 H, 15 N, 13 C EphB2 -KD was dissolved at a protein concentration of 1.0 mm in NMR buffer (20 mm HEPES buffer ph 7.2, 150 mm NaCl, 0.03% (w/v) NaN 3, and 2 mm DTT in 90% H 2 O / 10% D 2 O) for resonance assignment, while the 2 H, 15 N, 13 C phospho-ephb2 -KD was dissolved at ph 6.8 at a concentration of 0.8 mm. Chemical shift perturbation studies were performed on 2 H, 15 N-labled EphB2 fragments dissolved in NMR buffer at ph 7.2. Crystallization, Data Collection, and Structure Determination Hanging drops containing equal volumes of protein solution (EphB2 D754A KD at 16 mg/ml protein with 3.5 mm ADP and 10mM MgCl 2 ) and reservoir buffer containing 12% (w/v) PEG 20,000 and 0.1 M MES ph 6.5 resulted in crystals at 20ºC of the space group P2 1 (a = Å, b = Å, c = Å, α = γ = 90º, β = 90.18º), with four molecules per asymmetric unit. Diffraction data were collected under cryogenic conditions to 2.6 Å using a Rigaku RU- H3R rotating anode X-ray generator with Osmic optics (λ = Å) and R-AXIS IV++ imaging plate. The data were processed with the HKL program suite and molecular replacement solutions determined with CNS using the active insulin receptor kinase domain (PDB ID 1IR3), with side chains changed to alanine, as a search model. EphB2 side chains were built into the electron density maps using the program O. The model was refined (CNS) to a working R factor of 20.4% and a free R factor of 26.1%. Pertinent statistics for data collection and refinement are shown in Table I. 3

4 Hanging drops containing equal volumes of protein solution (EphA4 Y/F, Y750A -KD fragment at 16 mg/ml protein with 3.5 mm ADP and 10mM MgCl 2 ) and reservoir buffer containing 10% (w/v) PEG 8000, 0.1 M Tris ph 7.0, resulted in crystals at 20 ºC of the space group P2 1 (a = Å, b = Å, c = Å, α = γ = 90º, β = 108.8º) 1 with one molecule per asymmetric unit. Diffraction data were collected and processed to 2.35 Å as above, and molecular replacement solutions determined with CNS using the KD of autoinhibited EphB2 (aa , PDB ID 1JPA) as a search model. The model was refined as described for EphB2 KD to a working R factor of 20.9% and a free R factor of 24.8%. No electron density for ADP was visible in the structure. Immobilized peptide array kinase assays High density peptide (13mer) arrays were synthesized on acid hardened cellulose membrane with a Multipep robot (Intavis) using standard 9-fluorenylmethoxycarbonyl (F-moc) chemistry (Frank, 1992). Membranes were first wet with EtOH then washed three times with TBS-T prior to blocking overnight in 5% BSA in TBS-T. The membranes were then washed three times with TBS-T, two times with TBS, and two times with KRB prior to incubating with 100 µg of a purified active WT EphA4 fragment (including the and the kinase domain aa ), 10 µm ATP, and 50 µci 32 P-γ-ATP for 2 hours at room temperature with gentle agitation. Three separate 20 minute washes in 8M Urea, 1% SDS, 0.5% β-mercaptoethanol were followed by a single 20 minute wash in 50% EtOH, 10% acetic acid. The membrane was wet with EtOH and allowed to air dry prior to autoradiography using Kodak Biomax XAR film. 4

5 Supplementary References Binns, K.L., Taylor, P.P., Sicheri, F., Pawson, T. and Holland, S.J. (2000) Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors. Mol. Cell. Biol., 20, Frank, R. (1992). Spots-synthesis: an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron, 48, Wybenga-Groot, L.E., Baskin, B., Ong, S.H., Tong, J., Pawson, T. and Sicheri, F. (2001) Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell, 106,

6 Supplementary Table and Figure Legends Table SI. N-lobe RMSD C-lobe RMSD Last ordered First ordered versus autoinhibited versus auto- N-terminal C-terminal EphB2 inhibited EphB2 residue of the residue of the (Y/F - (Y/F - activation activation KD) molecule A KD) molecule A segment b segment b (aa ) a (aa ) a EphB Å 2 (85 C α ) 0.21 Å 2 (165 C α ) Y/F -KD molecule B EphB2 KD 0.39 Å 2 (87 C α ) 0.27 Å 2 (166 C α ) molecule A EphB2 KD 0.52 Å 2 (87 C α ) 0.24 Å 2 (166 C α ) molecule B EphB2 KD 0.38 Å 2 (87 C α ) 0.29 Å 2 (166 C α ) molecule C EphB2 KD 0.77 Å 2 (87 C α ) 0.26 Å 2 (166 C α ) molecule D EphA Å 2 (84 C α ) 0.38 Å 2 (165 C α ) 778 (770) 795 (785) Y/F, Y750A - KD EphA2 KD 0.46 Å 2 (87 C α ) 0.44 Å 2 (166 C α ) 776 (760) 795 (778) molecule A EphA2 KD 0.70 Å 2 (87 C α ) 0.58 Å 2 (166 C α ) 780 (764) 795 (778) molecule B RMSD comparison of Eph receptor active state and auto-inhibited state structures. a The number of C α atoms used for superposition is given in parentheses. b For clarity, EphB2 numbering is employed with the corresponding EphA4 and EphA2 numbering in parentheses. 6

7 Supplementary Figure Legends Figure S1. Schematic of EphA4 full-length and EphA4 and EphB2 kinase domain fragments used in this study. Figure S2. Effect of Tyr750Glu mutation on EphA4 catalytic 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 equivalent quantities of EphA4 proteins were employed in each kinase reaction. (B) A histogram of the specific activities of purified EphA4 proteins measured using a spectrophotometric coupling assay with 0.5 mm S1 peptide and 0.5 µm EphA4 kinase protein. In both experiments an EphA4 fragment containing the SAM domain was used. However, we have previously shown that a WT EphA4 fragment containing the, KD and SAM domain is indistinguishable from a fragment lacking the SAM domain in terms of kinase activity (Binns et al., 2000). Figure S3. The EphA4 kinase phosphorylates tyrosine residues within the but not Y750 or Y act. Peptides containing, Y750, and Y act motifs (13mer sequences as shown) from mouse EphA2 (gi ), mouse EphA4 (gi ) and mouse EphB2 (gi ) were arrayed onto a solid membrane support by spots synthesis and subjected to an in vitro radioactive kinase assay using an active fragment of the mouse EphA4 kinase. For each peptide motif, two equivalent peptides were synthesized with either a tyrosine residue (left) or a phenylalanine residue (right) at the probed position (indicated in bold). The incorporation of radiolabelled ATP was visualized using autoradiography. Figure S4. Comparison of active state and auto-inhibited EphB2 KD Crystal Structures. 7

8 Stereo view of the inter-lobe cleft, highlighting the improved coordination of bound nucleotide by active state EphB2. The backbone of active state EphB2 is coloured light blue with G-loop in pink and bound ADP nucleotide in yellow. The backbone of auto-inhibited EphB2 is coloured dark blue with G-loop and the ordered base of the bound nucleotide AMP-PNP in orange. Note that the sugar and phosphate groups of AMP-PNP are disordered. The two structures were aligned using C α atoms of the C-terminal lobes. Figure S5. Unfolding of the upon phosphorylation of residues Tyr and Tyr. Differences of 13 C α and 13 C β secondary chemical shifts of the unphosphorylated (A) and phosphorylated (B) EphB2 -KD. (C) Difference of secondary chemical shift differences shown in (A) and (B) demonstrating that changes in secondary structure upon phosphorylation of Y/ are limited to the. In all panels, the and the activation segment are highlighted with dashed boxes. Secondary structure elements are indicated with gray boxes labeled on top. Secondary chemical shifts are > 2 ppm for a continuous stretch of residues in α-helical regions and < -2 ppm for β-sheets. Figure S6. Spectral perturbation upon EphB2 activation. Spectral perturbations were quantified as relative peak intensities by dividing peak intensities in the active state spectra with those in the auto-inhibited reference spectrum. Peak intensities were measured at the precise position of the assigned peak in the auto-inhibited EphB2 spectra. Relative peak intensities smaller than 0.4 were considered significant and mapped onto the structure of the auto-inhibited EphB2 -KD fragment in Figure 3. Residues exhibiting significant spectral overlap upon kinase activation (relative peak intensities > 1.4) were excluded from the analysis. 8

9 Figure S7. Phosphorylation of the EphB2 leads to dramatic spectral perturbations in the and KD. Overlay of the 1 H, 15 N-HSQC spectra of the auto-inhibited EphB2 - KD (black) and the EphB2 -KD phosphorylated on residues Y and Y (red). Chemical shift changes within the are indicated by red dotted lines, while chemical shift changes within the KD are shown by black dotted lines. Residues in the (labeled in bold) relocate from well-dispersed regions of the HSQC spectrum of the auto-inhibited EphB2 - KD to proton chemical shifts around 8.0 ppm. Residues in the catalytic loop and the activation segment are underlined and experience increased line-broadening upon kinase activation. 9

10 Supplementary Figure 1 Wiesner et al. EphA4 mutants Full-length / -KD wild-type N-lobe KD C-lobe Mutagenesis Full-length / -KD Y/F Full-length / -KD Y/F, Y750A -KD Y/F, Y750E Full-length Y/F, Y750F Full-length / -KD Y750F Full-length Y750A KD Mutagenesis 750 KD Mutagenesis, crystal structure 750 KD Mutagenesis 750 KD Mutagenesis 750 KD Mutagenesis 750 KD Mutagenesis KD Y/F, Y788F KD -KD Y/F, Y750A, Y788F KD Mutagenesis Mutagenesis EphB2 mutants N-lobe C-lobe -KD D754A 754 KD NMR studies -KD Y750A, D754A KD NMR studies -KD py/, D754A 754 KD NMR studies KD D754A 754 KD Crystal structure, NMR studies

11 Supplementary Figure 2 Wiesner et al. A WT EphA4 Y750E EphA4 phospho EphA4 Autoradiography EphA4 Coomassie Stain B Specific Activity (%) WT Y750E

12 Y589/Y595 Y589/F595 F589/Y595 Y589 EQLKPLKTYVDPH Y595 VDPHTYEDPNQAV LKTYVDPHTYEDP LKTYVDPHTFEDP LKTFVDPHTYEDP Y736 LANMNYVHRDLAA Y773 DDPEATYTTSGGK Y750 Yact EphA4 Y596 LNQGVRTYVDPFT Y602 VDPFTYEDPNQAV Y596/Y602 VRTYVDPFTYEDP Y596/F602 VRTYVDPFTFEDP F596/Y602 VRTFVDPFTYEDP Y742 LSDMSYVHRDLAA Y779 DDPEAAYTTRGGK Y750 Yact EphB2 Y MTPGMKIYIDPFT Y IDPFTYEDPNEAV Y/Y MKIYIDPFTYEDP Y/F MKIYIDPFTFEDP F/Y MKIFIDPFTYEDP Y750 LADMNYVHRDLAA Y788 DTSDPTYTSALGG Y750 Yact EQLKPLKTFVDPH VDPHTFEDPNQAV LKTFVDPHTFEDP LKTFVDPHTFEDP LKTFVDPHTFEDP LANMNFVHRDLAA DDPEATFTTSGGK LNQGVRTFVDPFT VDPFTFEDPNQAV VRTFVDPFTFEDP VRTFVDPFTFEDP VRTFVDPFTFEDP LSDMSFVHRDLAA DDPEAAFTTRGGK MTPGMKIFIDPFT IDPFTFEDPNEAV MKIFIDPFTFEDP MKIFIDPFTFEDP MKIFIDPFTFEDP LADMNFVHRDLAA DTSDPTFTSALGG Y F Y F Y F EphA2 F589/F595 F589/F595 F589/F595 F589 F595 F736 F773 F596 F602 F596/F602 F596/F602 F596/F602 F742 F779 F F F/F F/F F/F F750 F788 Supplementary Figure 3 Wiesner et al.

13 Supplementary Figure 4 Wiesner et al. EphB2 -KD Y/F Molecule A +AMP-PNP EphB2 KD Molecule A + ADP αc EphB2 -KD Y/F Molecule A +AMP-PNP EphB2 KD Molecule A + ADP αc G-loop G-loop Lys 661 Glu 678 Lys 661 Glu 678 ADP ADP

14 Supplementary Figure 5 A Wiesner et al. Juxtamembrane segment αa αb β1 β2 β3 αc β4 β5 αd αe β7 β8 Activation segment αef αf αg αh αi αj B Juxtamembrane segment β1 β2 β3 αc β4 β5 αd αe 13 α 13 β δ C - δ C [ppm] β7 β8 Activation segment αef αf αg αh αi αj 13 α 13 β δ C - δ C [ppm] 13 α 13 β δ C - δ C [ppm] 13 α 13 β δ C - δ C [ppm] C Y/ - py/ [ppm] Juxtamembrane segment αa αb β1 β2 β3 αc β4 β5 αd αe Y/ - py/ [ppm] β7 β8 Activation segment αef αf αg αh αi αj

15 Supplementary Figure 6 Wiesner et al. Juxtamembrane segment αa αb β1 β2 β3 αc β4 β5 αd αe Relative Peak Intensity ( I rel ) cut-off β7 β8 Activation segment αef αf αg αh αi αj Relative Peak Intensity ( I rel ) cut-off Residue Number I rel (EphB2 -KD / EphB2 -KD) I rel (EphB2 -KD / EphB2 KD) I rel (EphB2 -KD / EphB2 py/ -KD) I rel (EphB2 -KD / EphB2 Y750A -KD) Residues within 5 Å of -KD interface

16 Supplementary Figure 7 N [ppm] Wiesner et al H [ppm] 1