Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domain Jeffrey J Babon1, Shenggen Yao1, David P DeSouza1,*, Christopher F Harrison1,*, Louis J Fabri2, Edvards Liepinsh3, Sergio D Scrofani2, Manuel Baca1,† and Raymond S Norton1 Walter and Eliza Hall Institute, Parkville, Victoria, Australia Amrad Corporation Ltd, Richmond, Victoria, Australia Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden Keywords cytokine signalling; NMR; PEST sequence; SOCS Correspondence J J Babon, Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3050, Victoria, Australia Fax: +61 93470852 Tel: +61 93452451 E-mail: babon@wehi.edu.au Present addresses *School of Biochemistry, University of Melbourne, Parkville 3050, Australia; †Amrad Corporation Ltd, 576 Swan Street, Richmond 3121, Victoria, Australia (Received 31 July 2005, revised 22 September 2005, accepted 10 October 2005) doi:10.1111/j.1742-4658.2005.05010.x SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single a-helix The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding Cytokine signalling acts through membrane-bound, multisubunit receptor complexes that are phosphorylated by activated Janus kinases (JAKs), leading to subsequent activation and phosphorylation of members of the signal transduction and activators of transcription (STAT) family The duration of the signalling response is moderated by a classic negative feedback control mechanism involving members of the suppressors of cytokine signalling (SOCS) family (SOCS1–7) and cytokine-inducible SH2-containing protein (CIS) The SOCS family members share similar architecture, including an N-terminal region of varying size, a central SH2 domain and a C-terminal SOCS box [1] (Fig 1) The SOCS SH2 domains are responsible for binding to phosphorylated tyrosine residues on intracellular domains of the cytokine receptors and ⁄ or the JAKs Abbreviations CIS, cytokine-inducible SH2-containing protein; ESS, extended SH2 subdomain; IPTG, isopropyl thio-b-D-galactoside; ITC, isothermal titration calorimetry; JAK, Janus kinase; KIR, kinase inhibitory region; PtdIns, phosphatidylinositol; SOCS, suppressor of cytokine signalling; STAT, signal transduction and activator of transcription 6120 FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS J J Babon et al Domain characterization of SOCS3 N-terminal SH2 SOCS box P 45 185 socs1 socs2 socs4 socs5 socs6 socs7 P P socs3 225 P P P CIS Fig The suppressor of cytokine signalling (SOCS) family of proteins The eight members of the SOCS family [SOCS1–7 and cytokine-inducible SH2-containing protein (CIS)] are shown schematically All eight members of the SOCS family contain a C-terminal SOCS box (black), a central SH2 domain (dark grey) and an N-terminal domain of varying lengths (light grey) CIS also contains a small insertion of  60 residues between the SH2 domain and the SOCS box (light grey) The SH2 domain boundaries shown for SOCS3 are as identified in this study The position of potential PEST motifs in the SOCS family, as suggested by this work, are indicated by a boxed ‘P’; note that they are not shown to scale SOCS4–7 have much longer N-terminal domains than the other SOCS family members (300–400 residues), the dotted lines indicate that these regions are not drawn to scale The residue numbering refers to SOCS3 only themselves [2] They act therefore by directly blocking signal transduction or by interfering with STAT access to the phosphorylated receptor subunits SOCS3, in particular, has a 22-residue N-terminal segment, followed by a 12-residue kinase inhibitory region (KIR) Mutation of essential residues in the KIR, or its deletion, affects kinase inhibition without affecting phosphotyrosine binding [3,4] One model proposed to explain the KIR action is that it can mimic the activation loop found in kinases such as JAK2 and FGF receptor kinase [5] and prevent substrate access to the catalytic groove of the kinase [6] In support of this, the sequences of the SOCS3 and SOCS1 KIRs share some homology with that of the JAK1 and JAK2 activation loops [6] There is no structural information available for the KIR, but if this mechanism operates it implies that the KIR is an extended loop or unstructured Immediately following the KIR in SOCS3 is the extended SH2 subdomain (ESS), an 11-residue segment preceding the true SH2 domain, which can affect phosphotyrosine binding via an unknown mechanism [3] There is no direct structural information available for the ESS, but sequence analysis, modelling [7] and the slight homology shared between the ESS and similar regions on Stat1 [8] and Stat3b [9] suggest that it may consist of one or two a-helices The SH2 domain of SOCS3 is immediately downstream from the ESS The SH2 domain is a common motif, present in proteins capable of binding to phos- photyrosine residues It typically contains around 100 residues, and adopts a fold consisting of a central b-sheet flanked on each face with an a-helix The SH2 domain of murine SOCS3 has been mapped previously by sequence comparison to residues 46–142 [10], but mutagenesis experiments have shown that residues as far away as Leu182 are important for phosphotyrosine–peptide binding [3] There is therefore some uncertainty about the extent of the SH2 domain, depending on whether it is predicted by sequence homology or functional analysis In addition to their role in blocking the activation of downstream signalling intermediates, the SOCS proteins may also act by directing the degradation of bound signalling molecules [11] As the C-terminal SOCS box is capable of interacting with an E3–ubiquitin ligase complex by binding directly to elongins B and C [12], SOCS proteins can recruit bound signal transduction proteins, such as activated kinases or the cytokine receptors themselves, for proteasome-mediated degradation [11,13,14] Although there is no structural information on the SOCS box, sequence and functional homologies suggest that it will adopt a similar structure to the corresponding region in the VHL protein [15], which is also responsible for binding to elonginB ⁄ C Reports differ as to whether the interaction between elonginB ⁄ C and SOCS stabilizes [16,17] or destabilizes [12,18] the SOCS proteins themselves Unambiguous secondary structure assignment, whether by NMR or other spectroscopic techniques, can FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6121 Domain characterization of SOCS3 J J Babon et al be a powerful tool in determining domain architecture In this study we show that the N-terminal portion of the KIR of murine SOCS3 is unstructured, but the C-terminal half of the KIR and the entire ESS form one single a-helix In addition, we show that the SH2 domain is a 140-residue domain that contains a 35-residue unstructured PEST motif insertion which is not required for phosphotyrosine binding but may have an important functional role Results hydrophilic in PtdIns 3-kinase but not in SOCS3 were identified and mutated to match the PtdIns 3-kinase residue The six mutants (A50D, G53R, L58E, A62E, A65E, G99D) were all cloned and expressed in E coli as part of a 22–185 construct All six constructs again expressed in inclusion bodies and required refolding The maximum concentration obtained by any of the six point mutants was  mgỈmL)1, in the presence of peptide, no higher than the wild-type SOCS3(22–185) construct As SOCS3(22–225) was too poorly soluble to obtain any meaningful structural data, the wild-type SOCS3(22–185) construct was pursued SOCS3 phosphotyrosine peptide complex Two initial constructs of mouse SOCS3 [SOCS3(22– 225) and SOCS3(22–185)] were cloned and expressed in Escherichia coli Both contain the KIR and the extended SH2 domain, but SOCS3(22–185) lacks the C-terminal SOCS box Both constructs expressed in inclusion bodies in E coli and required refolding A phosphotyrosine peptide from gp130 (STASTVEpYSTVVHSG) has been shown previously to bind with high affinity to mouse SOCS3 [19,20] The addition of a molar excess of peptide significantly increased < the solubility in NaCl ⁄ Pi from < mgỈmL)1 to  mgỈmL)1 for SOCS3(22–225) and to mgỈmL)1 for SOCS3(22–185) As SOCS3(22–185) in the presence of the tyrosinephosphorylated peptide could not be concentrated beyond  0.2 mm, seven constructs of shorter length were expressed in E coli and their solubility examined All constructs contained the SH2 domain, as defined by sequence homology [10], but included differing lengths of sequence outside this region All seven constructs (22–142, 22–128, 22–126, 44–185, 44–142, 44–128 and 44–126), and the control 22–185 and 22–225 fragments, were expressed in inclusion bodies in E coli and required refolding The construct showing the highest solubility was SOCS3(22–185) Constructs shorter than this at the C-terminal end did not bind tightly to the gp130 peptide (data not shown) All of the other constructs had equal or lower solubility, even in the presence of the tyrosine-phosphorylated peptide, including the predicted SH2 domain alone (44–142) This implied that the SH2 domain itself was a cause of poor solubility, as was the SOCS box The sequences of the SOCS3 SH2 domain and the phosphatidylinositol (PtdIns) 3-kinase (N-terminal) SH2 domain (the SH2 domain with the highest sequence identity in the PDB) were therefore aligned and hydrophobic residue substitutions in SOCS3 that were surface-exposed in the PtdIns 3-kinase structure were considered as candidates for point mutagenesis Six residues that were solvent-exposed and 6122 NMR assignments for murine SOCS3(22–185) After buffer optimization, SOCS3(22–185) was soluble to  0.5 mm, but UV-visible spectra of the protein showed that significant aggregation was occurring at this concentration, indicated by a high apparent absorption at 320 nm as a result of scattering Many NMR experiments required for full protein assignment therefore did not yield acceptable results, in particular HNCACB, HCCH-TOCSY and 13C-NOESY-HSQC Nevertheless, near-complete backbone resonance assignments were made for SOCS3(22–185) Apart from five missing spin systems (Ser25–Ser28 and Gly170), 100% of 1HN, 100% of 15N (excluding 18 proline residues), 96% of 13Ca, 84% of 13Cb, 87% of 13C¢ and 84% of 1Ha were assigned unambiguously (Fig 2) HNCO experiments were used to obtain 13C¢ resonances and therefore all 13C¢ N-terminal to proline residues remain unassigned The majority of side-chain assignments were determined, but because of the poor spectral quality of HCCH-TOCSY and 13C NOESY-HSQC experiments, no hydrophilic or polar c, d or e carbon assignments were made Secondary structure elements were determined by analysis of backbone and 13Cb chemical shifts (supplementary figure Fig S1), from characteristic NOE patterns in the 15N-edited NOESY-HSQC and by using talos [21] Assignments revealed that SOCS3 had an aabbbbbabbb topology, with the ESS and the C-terminal end of KIR forming the first a-helix (Fig 2) Significantly, there was a large unstructured region between Met128 and Arg163 that contained a high proportion of proline residues (12 out of 35) The chemical shifts of mouse SOCS3 have been deposited in BioMagRes-Bank (http://www.bmrb.wisc.edu) with accession number 6580 Murine SOCS3 contains a PEST region The sequence of the unstructured region of murine SOCS3 is highly conserved in mammalian SOCS3, as FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS J J Babon et al A Domain characterization of SOCS3 shown in Fig This region displays all of the common features of PEST sequences [22], namely a high proportion of Pro, Glu, Ser and Thr residues, the absence of Lys, His and Arg except at the termini, and the fact that it is completely unstructured based on the absence of medium- and long-range NOEs and the observation of intense backbone amide peaks (Fig S1) The primary sequence of SOCS3 was analysed for the presence of a PEST sequence by using the pestfind program (http://www.at.embnet.org/embnet/tools/bio/ PESTfind) [23] This analysis identified the likely presence (PESTfind score +11.11 [23]) of a single PEST sequence in SOCS3 spanning residues His126–Lys162 The unstructured region of SOCS3 spans Met128– Arg163 and therefore matches almost exactly the predicted PEST region Residues from Met128–Arg163 showed no inter-residue NOEs other than sequential connectivities, did not have restrained / ⁄ w angles according to TALOS, had amide resonances in the random coil region of the 15N-HSQC spectrum and showed significantly narrower line-widths than any other residues in the protein This indicates that the PEST sequence is an unstructured, highly mobile region within SOCS3 The PEST sequence is an insertion in the SH2 domain B Fig 15N-1H HSQC spectrum and secondary structure assignment of SOCS3(22–185) (A) The 15N-1H HSQC spectrum is shown of 0.1 mM SOCS3 at 500 MHz and 298 K in 50 mM sodium-phosphate buffer (pH 6.7) containing mM dithiothreitol The assigned residues are labelled with their residue number in the HSQC; some assignment labels are omitted for clarity (B) The secondary structure of SOCS3 was assigned by examining NOE patterns, analyses of backbone and 13Cb chemical shifts, and TALOS predictions [26], and is shown schematically with residue numbers marking the boundaries of each motif The PEST motif is shown as a thick black line The relevant secondary structure motifs are indicated at the top of the figure with the nomenclature used by Grucza et al [30] The topology of the b-sheet and two b-hairpins was determined by examining long-range backbone–backbone NOEs (supplementary table Table S1) ESS, extended SH2 subdomain; PEST, PEST motif Analysis of the secondary structure of SOCS3, and sequence alignments with SH2 domains, reveal that the PEST sequence begins immediately after the last residue of helix B in the SH2 domain However, most SH2 domains not end with this helix, but contain further structural elements at their C termini, including the ‘BG loop’ and the ‘G’-strand (Fig 2) [24] Hortner et al [25] have modelled the structure of the SOCS3 SH2 domain and suggest that the BG loop and bG strand are formed from residues Gly132–Val148, which we have shown to be unstructured and part of the PEST region We examined the sequence of the 19 structured residues immediately downstream of the PEST region and found a high likelihood that they constitute the BG loop and bG strand of the SH2 domain of SOCS3 (supplementary figure Fig S2) In particular, Leu176–Leu182 aligned well with the seven C-terminal residues of a number of SH2 domains, supporting this hypothesis In agreement with this scenario, deletion of residues 182–185 had been shown previously to affect phosphotyrosine peptide binding [3] Although the sequence between Tyr165 and Pro175, which would form the ‘BG loop’, was not significantly similar to other SH2 domains, the SHP-2 [26], grb7 [27] and, in particular, STAT3b [9] SH2 domains contain extended loops in this region that FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6123 Domain characterization of SOCS3 J J Babon et al Fig PEST sequence conservation in SOCS3 Sequence alignment of the region of SOCS3 containing the PEST motif for a number of mammalian species is shown, with conserved residues in the unstructured PEST motif shown hatched in grey The numbering refers to mouse SOCS3 The unstructured residues defined by this study are shown in bold structurally resemble a b-hairpin Analysis of the secondary structure of SOCS3 shows that it forms a b-hairpin in this region Thus, it appears that the PEST sequence constitutes an insertion in the true SOCS3 SH2 domain Murine SOCS3(D129–163) binds to a phosphotyrosine peptide from gp130 In order to determine whether deleting the PEST region would have an impact on the function of the SH2 domain, binding studies and isothermal titration calorimetry (ITC) were performed using a 22–185 construct lacking the PEST region [SOCS3(22–185)(D129– 163)] and the tyrosine phosphorylated peptide from gp130 SOCS3(D129–163) was constructed by replacing Pro129–Arg163 inclusive, with an eight residue [(Gly– Ser) · 4] linker in the 22–185 construct As shown in Fig 4, the construct lacking the PEST region binds to the gp130 peptide ITC analyses showed that the titration curve could be fitted using a single binding site mode with a Kd of 74 ± nm The Kd of wild-type SOCS3(22–185) binding was 152 ± 25 nm PEST sequences in other SOCS family proteins In order to determine whether other members of the SOCS family contained PEST motifs, their sequences were analysed using the PESTfind algorithm [23] Of the eight members of the murine SOCS family, SOCS1, -3, -5 and -7, and CIS, show a probable PEST motif with a PESTfind score of > (Table 1) CIS and SOCS3 have the PEST motif within the SH2 domain, while SOCS1, -5 and -7 contain PEST motifs in the N-terminal domain The PEST sequence in the CIS SH2 domain is located eight residues downstream from the terminus of the predicted aB helix Whether those eight residues are also unstructured, thus placing the unstructured insertion at an identical position to the PEST sequence in SOCS3, could not be determined Secondary structure prediction by sequence analysis gives no prediction for those eight residues 6124 The KIR ⁄ ESS consists of a single a-helix Based on observed NOEs, chemical shift deviations and TALOS predictions, residues Glu29–Ser44 form a single a-helix, whilst residues 22–28 are unstructured The helix encompasses the entire ESS and the four residues at the C terminus of the KIR The remaining residues that comprise the KIR appear to be unstructured Discussion In this study we defined the secondary structure elements of the SOCS3 protein, apart from the first 21 residues and the SOCS box The true SH2 domain boundaries were also defined for the first time, and an unstructured insertion therein was identified Residues 29–128 and 164–185 of SOCS3 are structured, but the N-terminal half of the KIR, and 35 residues in the SH2 domain, were shown by NMR to be unstructured This was evinced by the lack of any nonadjacent inter-residue NOEs in those regions, as well as the significantly sharper line-widths, characteristic of mobile, unstructured sections of polypeptide That the KIR is mostly unstructured when SOCS3 is in isolation is perhaps not surprising in view of the hypothesis for its mechanism proposed by Yasukawa et al [6] This requires the KIR to structurally mimic the activation loop of JAK2, so that in the absence of JAK2 the KIR would consist of an extended loop structure or be completely unstructured and separate from the globular core of the protein, so it can access, and block, the catalytic groove of the JAK2 kinase domain Mutagenesis studies have shown that a number of residues [3,4,25,28], important in binding phosphotyrosine-containing peptides or proteins, lie outside the SH2 domain predicted by sequence homology This led to the 12 residues immediately upstream of the SH2 domain being designated the ESS Our secondary structure assignment of SOCS3 shows that the entire ESS forms a single a-helix Giordanetto & Kroemer [7] modelled the structures of the ESS and KIR of FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS J J Babon et al Domain characterization of SOCS3 Fig SOCS3 lacking the PEST motif binds a gp130 peptide with high affinity (A) Titration of 80 lM gp130 peptide into 10 lM SOCS3(D101–133) The integrated heats from which the heat of dilution has been subtracted are shown, as well as the fit to a single site binding isotherm that yielded Kd 78 nM and DH )6.4 kcal mol)1 (B) Titration of 160 lM gp130 peptide into 13 lM wild type SOCS3 The fit to a single site binding isotherm is shown, yielding a Kd of 168 nM and a DH of )6.2 kcalỈmol)1 Both the wildtype and (D101-133) proteins used spanned residues 22–185 and not 22–225 because of the higher solubility of the former SOCS1, based on the similarity of the ESS sequence to a similar region in Stat1 [8] and Stat3b [9], and suggested that the ESS and KIR form two short orthogonal helices This differs from the single a-helix found in SOCS3, but a comparison of the ESS sequences from SOCS1 and SOCS3 shows that there are several divergent residues in this region, including a leucine (Leu32) in SOCS3 in place of an arginine (Arg67) in SOCS1, predicted in their model to make a critical ion pair with Asp76 The identification, by deletion mutagenesis, of residues affecting phosphotyrosine binding but located > 50 residues downstream of the predicted C terminus of the SH2 domain suggested that the functional SH2 domain was longer than originally suggested by sequence comparison [3] However, subsequent attempts to determine key residues important for the binding specificity of SOCS3 by structural modelling were hampered by the logical, yet incorrect, assumption that the SH2 domain consisted of c 100 contiguous residues In this report we have shown that the true SH2 domain is disrupted in murine SOCS3 by a 35-residue unstructured insertion that is predicted to form a PEST motif [22] This results in residues 164– 185 forming the BG loop and bG strand of a classic SH2 domain [24], rather than residues 129–147, as commonly assumed [25] This information is crucial for future attempts to alter the specificity of the SOCS3 SH2 domain by point mutation The PEST sequence identified in SOCS3 does not occur, according to sequence analysis, in the same location in any other members of the SOCS family, apart from CIS Analysis of the sequence of CIS using the PESTfind algorithm [23] shows a probable PEST sequence in residues 172–187, a region suggested by sequence homology to be located in a similar site in the SH2 domain as in SOCS3 Other members of the SOCS family contain putative PEST sequences, but these are all located in the N-terminal region, upstream of the SH2 domain The conservation of the PEST motif of SOCS3 in mammals (Fig 3), and the presence of probable PEST regions in most SOCS family members, suggests that it has an important functional role The appropriate duration of the cellular response to cytokine signalling will be determined, in large part, by the rate of turnover of the SOCS proteins Expression of the SOCS proteins is induced directly by STAT binding to the appropriate promoters Rapid destruction of the SOCS protein is also necessary, once signalling has ceased, to allow for subsequent cytokine stimulation The level of SOCS1 and SOCS3 protein in vivo appears to be strongly regulated by protein degradation, and the short half-life of SOCS proteins intracellularly appears to be the result primarily of proteolytic degradation [28,29] This may be important mechanistically, as the efficient turnover of SOCS proteins, and their induction of degradation of associated signalling molecules via the SOCS box, allows cells to respond to cytokine stimulation, quickly inhibit any prolonged activation and rapidly return to basal SOCS levels, ready for another round of stimulation There appear to be a number of features important for effective degradation of SOCS proteins, even apart from any role the SOCS box may play in this process Sasaki et al [28] have shown that a naturally occurring alternative transcript of SOCS3, lacking the first 11 FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6125 Domain characterization of SOCS3 J J Babon et al Table Predicted PEST motifs in suppressor of cytokine signalling (SOCS) family proteins Sequences of predicted PEST motifs in the SOCS family members are shown with their PESTfind score [29] and domain location NA indicates, for SOCS-2, -4 and -6, that no PEST motif was predicted for these proteins Protein SOCS1 SOCS2 SOCS3 SOCS4 SOCS5 SOCS6 SOCS7 CIS PESTfind score Sequence Location 14.2 NA 11.1 NA 7.4 11.3 NA 11.9 9.0 22 RSEPSSSSSSSSPAAPVR 39 NA 126 HYMPPPGTPSFSLPPTEPSSEVPEQPPAQALPGSTPK 162 NA 96 KDSDSGATPGTR 107 243 HSTFFDTFDPSLVSTEDEEDR 263 NA 74 KTAGGGCCP CPCPPQPPPPQPPPPAAAPQAGEDPTETSDALLVLEGLESEAESLETNSCSEEELSSPGR 142 172 RSDSPDPAPTPALPMSK 188 N terminus NA SH2 NA N terminus N terminus NA N terminus SH2 residues, has a prolonged half-life in Ba ⁄ F3 haemopoietic cells owing to the absence of Lys6, a major ubiquitination site of SOCS3 Chen et al [18] found that the N-terminal region of SOCS (upstream of the SH2 domain) contained a site for pim-1 kinase phosphorylation that significantly increased SOCS1 stability SOCS3 has also been shown to interact, and be phosphorylated by, pim-1 kinase, at an unknown site, which also confers stability [30] Proteasome-induced proteolysis is catalysed by the presence of regions of unstructured sequence in a protein [31] The PEST sequence is one such sequence commonly found in intracellular proteins of extremely short half-life [22,32] PEST sequences are hydrophilic, contain a high proportion of proline, glutamic acid, serine and threonine residues, and not contain lysine, arginine or histidine The X-ray structures of several proteins containing PEST sequences have been determined, but in each case the electron density of the PEST sequence is missing (e.g NF-jb [33] and ornithine decarboxylase [34]), presumably because this region is unstructured and mobile They can act in a modular manner, as transplanting PEST sequences from unstable proteins into stable proteins has been shown to reduce the half-life of the resulting chimaeras [32,35,36] The presence of a PEST sequence has been shown to be important in the proteolysis ⁄ degradation of a number of proteins with diverse functions, such as the glutamate receptor [37], proto-Dbl [38], and c-Fos [39] Biophysical characterization of the NF-jb PEST sequence [40] has shown it to be solvent-exposed and probably unstructured The PEST sequence in SOCS3 is located between two secondary structural elements, namely the aB helix and the BG loop In all SH2 domains these are located on the opposite face of the protein to the phosphotyrosine-binding site, so the PEST sequence is 6126 not expected to interfere with phosphotyrosine binding by the SH2 domain Indeed, replacing the entire 35-residue PEST sequence with GSGSGSGS had little effect upon binding a phosphorylated gp130 peptide, as shown by ITC In fact, the construct lacking the PEST motif bound slightly more tightly to the phosphotyrosine containing gp130 peptide than did wildtype SOCS3(22–185) Whether the twofold change in Kd is significant is difficult to determine as the construct lacking the PEST motif shows significantly less aggregation than wild-type SOCS3, which could alter the binding kinetics without representing a truly enhanced Kd The similarity of the two Kd values implies that the PEST sequence does not significantly affect phosphotyrosine binding Structurally, the PEST sequence is a benign insertion that may nevertheless play a critical functional role in regulating cellular SOCS3 levels Our identification of the secondary structure and correct domain boundaries of SOCS3 will enable manipulation of SOCS3 function by rational mutagenesis This includes mutagenesis of the PEST motif, either by complete removal or by point mutagenesis, to determine the effect it has on the biological function of SOCS3 in vivo, as well as mutagenesis of the SH2 domain to alter substrate specificity These approaches will allow a more thorough dissection of SOCS3 activity Experimental procedures Cloning and expression Fragments of mouse SOCS3 were subcloned by PCR into a ligation-independent cloning vector constructed by one of us (JJB) The vector encodes constructs with the leader sequence MASYHHHHHHDYDIPTTENLYFQGAHDGS, which consists primarily of a His6-tag and a TEV protease FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS J J Babon et al cleavage site For unlabelled protein, expression was performed in baffled flasks, with cells grown to an attenuance (D) at 600 nm of 0.6 in superbroth containing 50 lgỈmL)1 kanamycin Expression was induced with mm isopropyl thio-b-d-galactoside (IPTG) Cells were harvested, h after induction, by centrifugation (6200 g, °C, 30 min) For 15 N labelling, cells were grown to a D at 600 nm of 0.6 in Neidhardt’s medium [41] containing 1.0 gỈL)1 15NH4Cl as the sole nitrogen source For 15N ⁄ 13C-labelled samples, 1.0 gỈL)1 15NH4Cl and gỈL)1 13C glucose were the sole sources of nitrogen and carbon, respectively Cells were harvested h after IPTG induction by centrifugation (6200 g, °C, 30 min) All SOCS3 clones express as insoluble inclusion bodies Domain characterization of SOCS3 were screened again at pH 6.7 At this pH only the presence of 50 mm arginine plus 50 mm glutamate [43] yielded an increase in protein solubility, from to 10 mgỈmL)1, although UV-visible spectroscopy suggested that there was significant aggregation at that concentration SDS ⁄ PAGE analysis of the protein after storage revealed that disulphide bond-linked multimers formed slowly over time in the absence of a reducing agent In the presence of dithiothreitol and EDTA, the protein was stable at °C for at least months The final buffer conditions chosen for SOCS3 were therefore: 20 mm sodium phosphate, 20 mm NaCl, 50 mm arginine, 50 mm glutamate, mm dithiothreitol, mm EDTA, pH 6.5 Concentration to 10 mgỈmL)1 was performed using centrifugal concentration devices (Amicon Inc., Beverly, MA, USA) Protein purification and buffer screening Inclusion bodies were prepared via cell homogenization and centrifugation at 20 000 g, and solubilized using m guanidine hydrochloride The soluble inclusion body preparation was then purified using Ni-nitrilotriacetic acid resin (Qiagen, Valencia, CA, USA) Protein binding was performed at pH 8.0, washing at pH 6.3, and elution at pH 4.5 The eluted protein was quantified by absorbance at 280 nm, diluted to 0.1 mgỈmL)1, then refolded by extensive dialysis against 25 mm sodium phosphate, 50 mm sodium chloride, mm 2-mercaptoethanol, pH 6.7 The refolded protein was tested for correct conformation by binding an aliquot to a column with immobilized phosphorylated gp130 peptide (STASTVEpYSTVVHSG; pY ¼ phosphotyrosine [19,20]) The refolded protein was limited in its solubility, but addition of a 1.5· molar excess of the gp130derived phosphopeptide increased the solubility to c mgỈmL)1 As this concentration was still too low for structure determination by high-resolution NMR, a thorough screen of buffer conditions was undertaken in an attempt to improve the maximum solubility obtainable for SOCS3(22–185) The buffer screen was performed in microdrop format [42] and studied the pH range from to in 0.5 unit intervals, the salt concentration from to 500 mm in 50 mm intervals, and temperatures of 4, 25 and 37 °C Both constructs of SOCS3 showed highest solubility in buffers of low salt and high pH, and at low temperature The buffer conditions chosen for further additive screening were 20 mm Tris, pH 8.5, 20 mm NaCl, at 25 °C This starting condition was used to test the effect of 14 different additives, most at several concentrations The additive screen yielded promising results, and SOCS3 was shown to be soluble to  10 mgỈmL)1 ( 0.5 mm) in buffers containing > 10% glycerol, > 0.5 m non detergent sulfobetaine (NDSB), > 0.5 m trehalose or 50 mm arginine plus 50 mm glutamate [43] However, initial NMR analysis showed that under these conditions, many amide cross-peaks were missing from 15N HSQC spectra As the high pH was judged to be the cause of this, the most promising additives NMR spectroscopy Spectra were recorded at 298 K on a Bruker Avance 500 (using a cryoprobe), DRX-600, DMX-600 (using a cryoprobe) and Varian Unity INOVA 800 spectrometers Conventional 2D TOCSY and NOESY spectra were obtained using 2048 complex data points in the directly detected dimension and typically 200–400 t1 increments A TOCSY spin-lock time of 60 ms and a NOESY mixing time of 120 ms were used Spectra were processed using xwinnmr (Bruker AG, Karlsruhe, Germany) or nmr-pipe [44], and were analysed using xeasy (version 1.3.13) [45] or nmrdraw [44] Spectra were referenced to the H2O signal at 4.77 p.p.m (298 K) or a small impurity at 0.15 p.p.m Ca, Cb, Ha, C¢, and N chemical shifts were used in the program TALOS [21] to obtain backbone torsion angle predictions Sequence-specific resonance assignments for the backbone were accomplished using HNCA, HN(CO)CA, CBCA(CO)NH, HN(CA)CO and HNCO experiments [46] Side-chain assignments were accomplished by combining the data from the following experiments: 15N-edited TOCSY-HSQC and NOESY-HSQC, HCCH-TOCSY and HCCH-COSY [46] ITC Isothermal calorimetric titrations were performed using a Microcal omega VP-ITC (MicroCal Inc., Northampton, MA, USA) SOCS3(22–185) was dialysed against buffer (50 mm NaCl, 50 mm arginine, 50 mm glutamate, mm 2-mercaptoethanol, pH 6.7) and the dialysis buffer was used to dissolve the tyrosine-phosphorylated gp130 peptide Experiments were performed at 298 K Solutions of 10– 25 lm SOCS3 in the cell were titrated by injection of a total of 290 lL of 80–200 lm of the gp130 peptide The heat of dilution of the gp130 peptide into buffer was determined in control experiments and subtracted from the raw data of the binding experiment The data were analysed using the evaluation software, Microcal Origin, version FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6127 Domain characterization of SOCS3 J J Babon et al 5.0, provided by the manufacturer The binding curve fitted a single-site binding mode in all cases, and Kd values were determined from experiments repeated at least twice 11 Acknowledgements We thank Gottfried Otting for generously recording NMR spectra on SOCS3 We thank the Knut and Alice Wallenberg Foundation for the cryoprobe used to record NMR spectra at 600 MHz and access to the 800 MHz NMR spectrometer at Biovitrum AB 12 References Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, Sprigg NS, Starr R, Nicholson SE, Metcalf D & Nicola NA (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes Proc Natl Acad Sci USA 95, 114–119 Wormald S 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pulsed field gradients Prog Nuclear Magn Reson Spectrosc 34, 93–158 FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6129 Domain characterization of SOCS3 J J Babon et al Supplementary material The following supplementary material is available for this article online: Fig S1 Chemical shifts and peak intensities for mSOCS3(22–185) The chemical shift differences from random coil are shown for the (A) CO, (B) Ca, (C) Ha, and (D) Cb atoms of mSOCS3 plotted against residue number (D) The average chemical shift index values for each residue are shown (E) 1H-15N HSQC peak intensities (arbitrary units) for each backbone amide Peak intensity in HSQC experiments is correlated approximately with T2 relaxation times and hence flexibility Note that the PEST insertion displays abnormally large peak intensities, suggesting that it is truly flexible Fig S2 The SOCS3 SH2 domain sequence and secondary structure alignment As SOCS3 and SHP2 bind to the same site on the gp130 receptor [1–3], the N-terminal SH2 domain of SHP2 and its eight closest 6130 sequence neighbours were aligned with SOCS3 Residues shown in upper case are those conserved amongst SH2 domains according to the conserved domain database [4] The secondary structure of the N-terminal SH2 domain of SHP2, as determined by Lee et al [5], is shown above in black, with the structural elements labelled according to classical SH2 domain nomenclature The secondary structure of SOCS3, as determined in the present study, is shown below in grey For sequence comparison, the PEST region has been omitted and its site of omission shown with a black triangle Residues 176–183 in SOCS3 align well with the bG strand in SHP2 and other SH2 domains The BG loop in SOCS3 is a short b-hairpin rather than a true ‘loop’ region * Growth factor receptor binding protein Table S1 Backbone–backbone NOEs that allowed helix and b-sheet topology determination Pairs of residues with either NH–NH or NH–Ha NOEs are listed FEBS Journal 272 (2005) 6120–6130 ª 2005 The Authors Journal Compilation ª 2005 FEBS ... elements at their C termini, including the ‘BG loop’ and the ‘G’-strand (Fig 2) [24] Hortner et al [25] have modelled the structure of the SOCS3 SH2 domain and suggest that the BG loop and bG strand... SH2 domain (dark grey) and an N-terminal domain of varying lengths (light grey) CIS also contains a small insertion of  60 residues between the SH2 domain and the SOCS box (light grey) The SH2. .. unstructured insertion therein was identified Residues 29–128 and 164–185 of SOCS3 are structured, but the N-terminal half of the KIR, and 35 residues in the SH2 domain, were shown by NMR to be unstructured