SOCS3 binds specific receptor JAK complexes to control cytokine signaling by direct kinase inhibition SUPPLEMENTARY INFORMATION

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1 SOCS3 binds specific receptor JAK complexes to control cytokine signaling by direct kinase inhibition Nadia J. Kershaw 1,2, James M. Murphy 1,2, Nicholas P.D. Liau 1,2, Leila N. Varghese 1,2, Artem Laktyushin 1, Eden L. Whitlock 1, Isabelle S. Lucet 3, Nicos A. Nicola 1,2 and Jeffrey J. Babon 1,2 1 Walter and Eliza Hall Institute, Parkville, Australia 2 The University of Melbourne, Parkville, Australia. 3 Monash University, Clayton, Australia. Correspondence should be addressed to J.J.B (babon@wehi.edu.au) or N.A.N (nicola@wehi.edu.au) SUPPLEMENTARY INFORMATION Running title: Inhibition of JAK/STAT signaling by SOCS3

2 Supplementary Figure 1 Crystallisation of SOCS/JAK2/gp130 (a) Schematic of the JAK2 and SOCS3 domain architectures. The regions boxed in red are those crystallised in this study. (b) F obs -F calc simulated annealing omit map calculated from model in which all 4 copies of gp130 were removed. Electron density for the gp130 fragment is shown in green mesh contoured at 3. (c) B-factor sharpened 2F obs -F calc electron density map showing the SOCS3/gp130 interface contoured at 1.2. Important residues are shown labeled

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4 Supplementary Figure 2 SAXS analysis of SOCS JAK2 gp130 (a). The pairwise interatomic distance distributions (P(r)) were determined using GNOM (Svergun 1992) from the scattering profiles for apo Jak2 JH1 (green) and the Jak2 JH1:SOCS3 SH2:gp130 peptide complex (blue). The maximum particle dimension, Dmax, is elongated for the Jak2 JH1:SOCS3 SH2:gp130 peptide complex relative to apo Jak2 JH1. Using GNOM, the radius of gyration, Rg, was determined as 22.00±0.047 Å for apo Jak2 JH1 compared to that of the Jak2 JH1:SOCS3 SH2:gp130 peptide complex, 26.19±0.093 Å. (b). A Guinier plot of the Jak2 JH1:SOCS3 SH2:gp130 peptide complex scattering data is linear, illustrating that high molecular weight aggregates do not measurably contribute to scattering. (c,d) The experimental scattering curve measured for the Jak2 JH1:SOCS3 SH2:gp130 peptide complex (black) were overlaid with (c) the theoretical scattering curve calculated in CRYSOL from the X-ray crystal structure of the complex and (d) the fitted curve arising from DAMMIF ab initio bead modelling (both shown as red curves). The values for the fits shown in (c) and (d) are and 0.506, respectively, indicating an excellent correspondence between experimental data and models. (e) The other potential SOCS/JAK/gp130 trimer. There are two potential SOCS/JAK interfaces present in the asymmetric unit however only one of them is consistent with NMR data, mutagenesis and SAXS. The alternate model is shown in cartoon representation. SOCS3 as present in our final model is shown in semi-transparent surface representation for comparison (f) The experimental scattering curve measured for the Jak2 JH1:SOCS3 SH2:gp130 peptide complex (black) was overlaid with the theoretical scattering curve calculated in CRYSOL from the X-ray crystal structure of the other potential SOCS/JAK/gp130 trimer in the asymmetric unit. This fit is poor ( =2.7) compared to that shown in (c). (g) The molecular envelope of the JAK2/SOCS3/gp130 complex in solution calculated from SAXS data by performing 10 independent DAMMIF ab initio bead reconstructions (grey spheres) superimposed with the alternate complex crystal structure. Upper panel shown in the same orientation as panel e, left; lower panel, top view.

5 Supplementary Figure 3 Electron density of of the SOCS3/JAK2 interface (a) B-factor sharpened 2F obs -F calc electron density map showing the SOCS3/JAK2 interface contoured at 1.5. Important residues are shown labeled. (b) Comparison of the JAK2 structure in isolation (PDB: 2B7A 1 ) and in complex. (c) A comparison of JAK2 and JAK3 2 highlights the GQM motif and JAK insertion loop as the major conformational differences between the two molecules.

6 Supplementary Figure 4 Electron density of the kinase inhibitory region B-factor sharpened 2F obs -F calc electron density map showing the SOCS3/JAK2 interface (SOCS3 in foreground) contoured at 1.5. Important residues are shown labeled. The view shown is the same as Figure 3A.

7 Supplementary Figure 5. SOCS3 co-precipitates with activated and dephosphorylated JAK2JH1 with similar affinity. Dephosphorylated JAK2 JH1 was prepared by co-expressing it with the phosphatase PTP1B and then used in the co-precipitation experiment shown in Figure 4b. An aliquot of the gel samples shown in Figure 4b were run on a second SDS-PAGE gel and western blotted using an anti-jak2 antibody (left panel) and an anti-pjak2 (py1007/8) antibody (center panel). These blots were performed on the same membrane using 700nm and 800nm Infraredlabelled secondary antibodies and visualized using the odyssey system. An overlay of the 700 nm and 800 nm channels is shown on the right.

8 Supplementary Figure 6 SOCS3 inhibits JAK2 by blocking substrate binding. (a) Kinase inhibition assays performed with constructs of SOCS3 truncated at the N-terminal end of the KIR show that constructs lacking 1-3 residues only partially inhibit JAK2 activity. Left, the phosphorimage of kinase inhibition experiments using the standard STAT5b peptide (Y+8) as a substrate. Right, as for left but using a C-terminally truncated (Y+1) substrate. Inhibition of STAT5bY+1 by SOCS3 N22,23 is only 50% complete under saturating conditions. These results are quantified and plotted in Figure 5c. (b) Radioactive kinase assays show that constructs of SOCS3 with a tyrosine 1-3 residues upstream of the KIR are good substrates for JAK2. Reactions were performed for 1 minute (upper panels) and 2 minutes (lower panels) and then analyzed via SDS-PAGE (left) followed by autoradiography (right). Experiments performed in the absence of SOCS3 (-ve) are shown as a control. (c) As for (b) but SOCS3 F25A mutants are used as controls to show that tyrosines upstream of the KIR are only good substrates for JAK2 when forced into close proximity of the active site by the remainder of SOCS3. These results are the same as those shown in Figure 6a,b with the addition of the Coomassie stained gels from the same experiment to indicate loading.

9 Supplementary Figure 7. JAK2 can phosphorylate a Threonine residue incorporated upstream of the SOCS3 KIR. Three constructs of SOCS3, containing either a Threonine, Glycine or Tyrosine three residues upstream of the KIR were tested as substrates for JAK2 phosphorylation. Reactions were performed for 1 minute and 2 minutes and then analyzed via SDS-PAGE (upper) followed by autoradiography (lower). 1mM STAT5b peptide was included in the reaction to compare the relative efficiency of phosphorylation. SOCS3/elonginBC constructs were present at 10 M.

10 Supplementary Table 1: SAXS data analysis. SAXS data analysis. Data-collection parameters Instrument Australian Synchrotron SAXS/WAXS beamline Beam geometry Wavelength (Å) 120 micron point source, Å q range (Å-1)a to Å-1 Exposure time, Protein concentration, Temperature 2sec exposures, 5mg/mL protein via inline gel filtration chromatography, 27 C. Structural parameters I(0) (cm-1) [from P(r)] ± Rg (Å) [from P(r)] ± 0.09 Dmax (Å) 85 I(0) (cm-1) (from Guinier) ± Rg (Å) (from Guinier) ± 0.12 Theoretical envelope volume (Å- 71,300 3)b Experimentally determined 70,820 excluded volume (Å-3)c Software employed Primary data reduction SAXS15ID (Australian Synchrotron) Data processing PRIMUS, GNOM Ab initio analysis DAMMIF Validation and averaging DAMAVER Rigid-body modelling N/A Computation of model intensities CRYSOL Three-dimensional graphics Pymol representations a q is the magnitude of the scattering vector, which is related to the scattering angle (2θ) and the wavelength (λ) as follows: q = (4π/λ)sinθ b Envelope volume calculated from crystal structure using CRYSOL c Excluded volume calculated from 10 averaged DAMMIF ab initio bead models

11 Supplementary Note Crystal structure determination Initial building and refinement of the structure was performed using data cut at 4.5Å, using I/σI of 1.5 as the data cut-off criterion. The structure was refined to excellent R free and R work values of and respectively. The low R-factors are aided by the presence of 4-fold non-crystallographic symmetry and the fact that high resolution structures of SOCS3 (2.0Å) and JAK2 (2.4Å) made up the initial model. However, during the course of this work, Karplus and Diederichs published an alternative criterion for data cut-off 3. Using this method we extended the data cut-off to 3.9Å and were able to refine our model to even lower R free and R work values of and respectively with improvement in the electron density maps. R free and R work in the Å bin dropped by >3% when the additional data was included. The improvement in both resolution and refinement was likely due to the fact that the X-ray diffraction pattern was highly anisotropic and that, in some dimensions, diffraction could be observed out to 3.6Å. Refinement also required less stringent restraints when the additional data was included. As part of the refinement procedure, thermal factor sharpening (B-factor sharpening) was applied to 2F o -F c maps in order to gain more detailed information on atomic positions, especially those in amino acid side chains. The B-factor sharpening technique, which multiplies the structure factor of a particular electron density map by an exponential function of the negative Wilson B factor has been shown by Brunger and colleagues to be particularly effective during refinement of mid-low resolution datasets in the Å range where excellent phases already exist due to higher resolution initial models 4. As these conditions were met by our dataset we applied thermal factor sharpening cautiously during the final few stages of refinement and always compared the resulting maps with nonsharpened maps. This procedure aided us in obtaining excellent refinement statistics given our relatively low resolution dataset. For completeness, toward the end of refinement, we included the low twin fraction in our refinement. The twin law used, as determined by Xtriage, was h,-h-k,-l, and the twin fraction refined to There was no significant difference in maps generated with and without twinning. Final R free and R work values of and were obtained, as reported in Table 1. Supplementary References 1. Lucet, I.S. et al. The structural basis of Janus kinase 2 inhibition by a potent and specific pan-janus kinase inhibitor. Blood. 107, Epub 2005 Sep 20. (2006). 2. Boggon, T.J., Li, Y., Manley, P.W. & Eck, M.J. Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog. Blood 106, (2005). 3. Karplus, P.A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, (2012). 4. Brunger, A.T., DeLaBarre, B., Davies, J.M. & Weis, W.I. X-ray structure determination at low resolution. Acta Crystallogr D Biol Crystallogr 65, (2009).