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 r
Trang 1defines 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
1 Walter and Eliza Hall Institute, Parkville, Victoria, Australia
2 Amrad Corporation Ltd, Richmond, Victoria, Australia
3 Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
Cytokine signalling acts through membrane-bound,
multisubunit receptor complexes that are
phosphoryl-ated by activphosphoryl-ated Janus kinases (JAKs), leading to
sub-sequent 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, inclu-ding 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
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 3 93470852
Tel: +61 3 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 pro-tein, cytokine-inducible SH2-containing propro-tein, 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 bind-ing, 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 down-stream than predicted by sequence homology, explains a number of previ-ously unexpected results that have shown the importance of residues close
to the SOCS box for phosphotyrosine binding
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.
Trang 2themselves [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
sub-strate 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
phos-photyrosine 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
down-stream 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 phosphotyro-sine–peptide binding [3] There is therefore some uncer-tainty 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 pro-teins 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–ubiqu-itin 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-medi-ated degradation [11,13,14] Although there is no struc-tural information on the SOCS box, sequence and functional homologies suggest that it will adopt a sim-ilar 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 interac-tion between elonginB⁄ C and SOCS stabilizes [16,17]
or destabilizes [12,18] the SOCS proteins themselves Unambiguous secondary structure assignment, whe-ther by NMR or owhe-ther spectroscopic techniques, can
socs1 socs2 socs3
socs4 socs5 socs6 socs7
CIS
N-terminal SH2 SOCS
box
P
P
P P
P P
Fig 1 The suppressor of cytokine signalling (SOCS) family of proteins The eight members of the SOCS family [SOCS1–7 and cytokine-indu-cible 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.
Trang 3be 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
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
(STASTV-EpYSTVVHSG) has been shown previously to bind
with high affinity to mouse SOCS3 [19,20] The
addi-tion of a molar excess of peptide significantly increased
the solubility in NaCl⁄ Pi from << 1 mgÆmL)1 to
1 mgÆmL)1 for SOCS3(22–225) and to 3 mgÆmL)1
for SOCS3(22–185)
As SOCS3(22–185) in the presence of the
tyrosine-phosphorylated 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
con-structs had equal or lower solubility, even in the
pres-ence 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
solubil-ity, 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-kin-ase structure were considered as candidates for point
mutagenesis Six residues that were solvent-exposed and
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 3 mgÆmL)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
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 absorp-tion at 320 nm as a result of scattering Many NMR experiments required for full protein assignment there-fore did not yield acceptable results, in particular HNCACB, HCCH-TOCSY and 13C-NOESY-HSQC Nevertheless, near-complete backbone resonance assign-ments 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% of13Ca, 84% of13Cb, 87% of13C¢ and 84% of1Ha were assigned unambiguously (Fig 2) HNCO experi-ments were used to obtain13C¢ resonances and therefore all 13C¢ N-terminal to proline residues remain unas-signed 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 deter-mined by analysis of backbone and13Cbchemical shifts (supplementary figure Fig S1), from characteristic NOE patterns in the15N-edited NOESY-HSQC and by using talos [21] Assignments revealed that SOCS3 had
an aabbbbbabbb topology, with the ESS and the C-ter-minal end of KIR forming the first a-helix (Fig 2) Sig-nificantly, 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
Trang 4shown in Fig 3 This region displays all of the com-mon 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 pres-ence (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 pre-dicted 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
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 do 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 exam-ined the sequence of the 19 structured residues immedi-ately 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 pep-tide 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
A
B
Fig 2 15N- 1 H HSQC spectrum and secondary structure
assign-ment of SOCS3(22–185) (A) The 15 N- 1 H HSQC spectrum is shown
of 0.1 m M SOCS3 at 500 MHz and 298 K in 50 m M
sodium-phos-phate buffer (pH 6.7) containing 2 m M dithiothreitol The assigned
residues are labelled with their residue number in the HSQC; some
assignment labels are omitted for clarity (B) The secondary
struc-ture of SOCS3 was assigned by examining NOE patterns, analyses
of backbone and 13 Cbchemical 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.
Trang 5structurally resemble a b-hairpin Analysis of the
secon-dary 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
con-struct 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
titra-tion curve could be fitted using a single binding site
mode with a Kd of 74 ± 7 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 > 5 (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
unstruc-tured, thus placing the unstructured insertion at an
identical position to the PEST sequence in SOCS3,
could not be determined Secondary structure
predic-tion by sequence analysis gives no predicpredic-tion for
those eight residues
The KIR⁄ ESS consists of a single a-helix Based on observed NOEs, chemical shift deviations and TALOS predictions, residues Glu29–Ser44 form a sin-gle 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 ele-ments 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 unstruc-tured This was evinced by the lack of any nonadja-cent 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 isola-tion 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 activa-tion loop of JAK2, so that in the absence of JAK2 the KIR would consist of an extended loop struc-ture or be completely unstrucstruc-tured 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 phosphotyro-sine-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
Fig 3 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.
Trang 6SOCS1, based on the similarity of the ESS sequence to
a similar region in Stat1 [8] and Stat3b [9], and
sugges-ted 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
diver-gent 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
resi-dues 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,
assump-tion that the SH2 domain consisted of c 100
contigu-ous 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 mem-bers, 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 destruc-tion of the SOCS protein is also necessary, once signal-ling has ceased, to allow for subsequent cytokine stimulation The level of SOCS1 and SOCS3 protein
in vivo appears to be strongly regulated by protein de-gradation, 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 pro-teins, 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 effect-ive 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
Fig 4 SOCS3 lacking the PEST motif binds
a gp130 peptide with high affinity (A)
Titra-tion of 80 l M gp130 peptide into 10 l M
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 n M and DH )6.4 kcal mol )1 (B) Titration
of 160 l M gp130 peptide into 13 l M wild
type SOCS3 The fit to a single site binding
isotherm is shown, yielding a K d of 168 n M
and a DH of )6.2 kcalÆmol )1 Both the
wild-type and (D101-133) proteins used spanned
residues 22–185 and not 22–225 because of
the higher solubility of the former.
Trang 7residues, has a prolonged half-life in Ba⁄ F3
haemopoi-etic cells owing to the absence of Lys6, a major
ubiqui-tination site of SOCS3 Chen et al [18] found that the
N-terminal region of SOCS 1 (upstream of the SH2
domain) contained a site for pim-1 kinase
phosphory-lation that significantly increased SOCS1 stability
SOCS3 has also been shown to interact, and be
phos-phorylated 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
pro-tein [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 do 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
ornith-ine decarboxylase [34]), presumably because this region
is unstructured and mobile They can act in a modular
manner, as transplanting PEST sequences from
unsta-ble proteins into staunsta-ble 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
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 phos-photyrosine containing gp130 peptide than did wild-type SOCS3(22–185) Whether the twofold change in
Kd is significant is difficult to determine as the con-struct 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 cor-rect domain boundaries of SOCS3 will enable manipu-lation of SOCS3 function by rational mutagenesis This includes mutagenesis of the PEST motif, either by com-plete 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
Table 1 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
PESTfind
SOCS7 11.9 74 KTAGGGCCP CPCPPQPPPPQPPPPAAAPQAGEDPTETSDALLVLEGLESEAESLETNSCSEEELSSPGR 142 N terminus
Trang 8cleavage site For unlabelled protein, expression was
per-formed in baffled flasks, with cells grown to an attenuance
(D) at 600 nm of 0.6 in superbroth containing 50 lgÆmL)1
kanamycin Expression was induced with 1 mm isopropyl
thio-b-d-galactoside (IPTG) Cells were harvested, 3 h after
induction, by centrifugation (6200 g, 4C, 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 gÆL)1 15NH4Cl as
the sole nitrogen source For 15N⁄13
C-labelled samples, 1.0 gÆL)1 15NH4Cl and 2 gÆL)1 13C glucose were the sole
sources of nitrogen and carbon, respectively Cells were
harvested 8 h after IPTG induction by centrifugation
(6200 g, 4C, 30 min) All SOCS3 clones express as
insol-uble inclusion bodies
Protein purification and buffer screening
Inclusion bodies were prepared via cell homogenization and
centrifugation at 20 000 g, and solubilized using 6 m
guani-dine hydrochloride The soluble inclusion body preparation
was then purified using Ni-nitrilotriacetic acid resin
(Qiagen, Valencia, CA, USA) Protein binding was
per-formed 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 mgÆmL)1, then refolded by extensive
dialysis against 25 mm sodium phosphate, 50 mm sodium
chloride, 5 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; pY¼
phospho-tyrosine [19,20]) The refolded protein was limited in its
solubility, but addition of a 1.5· molar excess of the
gp130-derived phosphopeptide increased the solubility to
c 3 mgÆmL)1 As this concentration was still too low for
structure determination by high-resolution NMR, a
thor-ough 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
micro-drop format [42] and studied the pH range from 4 to 9 in
0.5 unit intervals, the salt concentration from 0 to 500 mm
in 50 mm intervals, and temperatures of 4, 25 and 37C
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 25C 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 mgÆmL)1 ( 0.5 mm) in buffers
contain-ing > 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
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 3 to
10 mgÆmL)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 4C for
at least 2 months The final buffer conditions chosen for SOCS3 were therefore: 20 mm sodium phosphate, 20 mm NaCl, 50 mm arginine, 50 mm glutamate, 2 mm dithio-threitol, 1 mm EDTA, pH 6.5 Concentration to
10 mgÆmL)1 was performed using centrifugal concentration devices (Amicon Inc., Beverly, MA, USA)
NMR spectroscopy Spectra were recorded at 298 K on a Bruker Avance 500 (using a cryoprobe), DRX-600, DMX-600 (using a cryo-probe) and Varian Unity INOVA 800 spectrometers Con-ventional 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 pre-dictions 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, 5 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 deter-mined in control experiments and subtracted from the raw data of the binding experiment The data were analysed using the evaluation software, Microcal Origin, version
Trang 95.0, 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
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
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