https://doi.org/10.1007/s12104-020-09941-y ARTICLE
1
H,
13C,
15N chemical shift assignments of SHP2 SH2 domains in complex with PD‑1 immune‑tyrosine motifs
Michelangelo Marasco1 · John P. Kirkpatrick1,2 · Teresa Carlomagno1,2
Received: 11 February 2020 / Accepted: 26 March 2020 / Published online: 1 April 2020
© The Author(s) 2020
Abstract
Inhibition of immune checkpoint receptor Programmed Death-1 (PD-1) via monoclonal antibodies is an established anticancer immunotherapeutic approach. This treatment has been largely successful; however, its high cost demands equally effective, more affordable alternatives. To date, the development of drugs targeting downstream players in the PD-1-dependent signaling pathway has been hampered by our poor understanding of the molecular details of the intermolecular interactions involved in the pathway. Activation of PD-1 leads to phosphorylation of two signaling motifs located in its cytoplasmic domain, the immune tyrosine inhibitory motif (ITIM) and immune tyrosine switch motif (ITSM), which recruit and activate protein tyrosine phosphatase SHP2. This interaction is mediated by the two Src homology 2 (SH2) domains of SHP2, termed N-SH2 and C-SH2, which recognize phosphotyrosines pY223 and pY248 of ITIM and ITSM, respectively. SHP2 then propagates the inhibitory signal, ultimately leading to suppression of T cell functionality. In order to facilitate mechanistic structural studies of this signaling pathway, we report the resonance assignments of the complexes formed by the signaling motifs of PD-1 and the SH2 domains of SHP2.
Keywords PD-1 · SHP2 · Immunotherapy · SH2 domains
Biological context
The recent success of anticancer therapies that target immune checkpoint receptor PD-1 has sparked consider- able interest in the molecular details behind its signaling function. PD-1 is a 288-amino-acid receptor of the CD28 family, mostly expressed on the surface of T lymphocytes, whose main function is to maintain immune tolerance and prevent overactive T cell responses (Boussiotis 2016).
However, PD-1 functionalities are also exploited by certain cancer types to evade immune surveillance; for this reason, monoclonal antibodies that block the interaction between this receptor and its activation ligand PD-L1 have proven successful in the treatment of tumors such as metastatic
melanoma, non-small cell lung cancer and renal cell car- cinoma (Page et al. 2014; Topalian et al. 2015). Despite their efficacy, the very high cost of immunotherapies poses a severe burden on public healthcare systems and calls for novel, equally effective, but more affordable drugs (Prasad et al. 2017).
Targeting the PD-1-dependent signaling pathway has been made problematic by our limited knowledge of the molecular events following PD-1 activation; only recently have ground-breaking studies elucidated important mecha- nistic aspects of the PD-1-dependent signaling events. Acti- vation of PD-1 leads to phosphorylation of two key tyrosine residues in its cytoplasmic domain by Src family kinases (Sharpe and Pauken 2018; Hui et al. 2017). These two tyros- ines, Y223 and Y248, are embedded into two immune-tyros- ine signaling motifs, which are unique among the members of the CD28 family, namely the immune-tyrosine inhibitory motif (ITIM) and immune-tyrosine switch motif (ITSM), respectively (Riley 2009). The two phosphotyrosines recruit and activate Src homology 2 (SH2) domain-containing phos- phatase 2 (SHP2), which propagates the signal from PD-1 by dephosphorylating key tyrosines on CD28 (Hui et al. 2017).
* Teresa Carlomagno
teresa.carlomagno@oci.uni-hannover.de
1 Center for Biomolecular Drug Design and Institute of Organic Chemistry, Leibniz University Hannover, Schneiderberg 38, 30167 Hannover, Germany
2 Helmholtz Center for Infection Research, Group of NMR-Based Structural Chemistry, Inhoffenstrasse 7, 38124 Braunschweig, Germany
SHP2 is a 70-kDa protein of the PTP (protein tyrosine phosphatase) superfamily, which consists of three folded domains (two SH2 domains arranged in tandem, termed N-SH2 and C-SH2, and a catalytic PTP domain) followed by a disordered C-terminal tail with putative regulatory functions (Neel et al. 2003). In its basal state, SHP2 activity is very low, due to an auto-inhibitory interac- tion between the N-SH2 and the PTP, which occludes the catalytic site and prevents substrate processing (Hof et al.
1998). Engagement of the N-SH2 or both the N-SH2 and the C-SH2 by monovalent or divalent phosphopeptides, respectively, leads to the transition to an open conforma- tion, in which the N-SH2 is displaced and the catalytic site becomes accessible (Barford and Neel 1998). Muta- tions that disrupt the interaction between the N-SH2 and the PTP also favor the open conformation and have been associated with several diseases (Keilhack et al. 2005).
SH2 domains are protein modules of around 100 amino acids, with a conserved fold consisting of a cen- tral three-stranded antiparallel β sheet flanked by two α helices, whose function is to recognize phosphotyrosine- containing peptides (Waksman et al. 2004). In this work, the nomenclature for SH2–phosphopeptide complexes fol- lows the convention introduced by Eck and coworkers (Eck et al. 1993): the SH2 helices are named αA and αB, the β strands are βA–βG and the loops are defined based on the structural elements that they connect; phosphopeptide residues are numbered according to their position relative to the phosphotyrosine (pY–2, pY–1, pY+1, pY+2…).
The structural and biochemical details of the activa- tion of SHP2 by PD-1 derived phosphopeptides have been reported recently (Hui et al. 2017; Peled et al. 2018; Mar- asco et al. 2020). Interestingly, a peptide containing both ITIM and ITSM linked together is required for maximal stimulation of phosphatase activity. This bidentate pep- tide binds the N-SH2 and C-SH2 domains of one SHP2 molecule with ITIM and ITSM, respectively, to form a 1:1 doubly bound heterodimeric complex. This dual binding event requires a large rearrangement of the orientation of the SH2 domains in SHP2 in order to satisfy the spatial restraints imposed by the linker between ITIM and ITSM.
Therefore, formation of the 1:1 doubly bound complex is associated with overcoming a high conformational energy barrier, which slows down the association of the second SH2 domain with the second pY motif of the same peptide both in vitro and in vivo (Marasco et al. 2020; Oh et al.
2012). Consequently, the stoichiometry and oligomeric state of the complexes present in a mixture of SHP2 and bidentate peptide is heterogeneous, particularly in regimes of high protein concentrations or receptor clustering, with variable amounts of higher-order oligomeric protein–pep- tide complexes (wherein the SH2 domains of a single protein molecule engage pY motifs of different peptide
molecules) in addition to the 1:1 doubly-bound heterodi- mer (Marasco et al. 2020; Oh et al. 2012).
Here, we report the backbone resonance assignments for the unbound, ITIM-bound and ITSM-bound states of the N-SH2 and C-SH2 domains. In addition, we report the assignment of the protein side-chains and bound peptide resonances of the N-SH2–ITIM complex; the correspond- ing resonance assignment for the C-SH2–ITSM complex has been published previously (Marasco et al. 2020). These results have aided the assignment of the backbone reso- nances of tSH2 (a construct containing both N-SH2 and C-SH2 domains) in complex with the bidentate peptide ITIM-[dPEG4]2-ITSM, which contains both ITIM and ITSM joined by a polyethyleneglycol-based linker. The assignment of the resonances of the tSH2 and their shifts upon titration with the bidentate peptide revealed the presence of a het- erogeneous mixture of 1:1 heterodimers and higher-order protein–peptide oligomers at variable stoichiometric ratios (Marasco et al. 2020).
Methods and experiments
Protein expression and purificationThe DNA sequences of human N-SH2 (SHP21−105), C-SH2 (SHP2106−220) and tSH2 (SHP21−220) were cloned into the pETM22 expression vector, which allows for expression of the target proteins as fusion constructs with a cleavable His6-thioredoxin tag. The vectors were transformed into Tuner (DE3) competent cells (Merck). For recombinant protein expression, freshly transformed cells were grown at 37 °C to an optical density (OD) of 0.6–0.8. Afterwards, the culture was quickly chilled and 0.1 mM of isopropyl β-d-1- thiogalactopyranoside (IPTG) was added to induce protein expression, which was continued for 20 h at 20 °C. Prepara- tion of U-13C,15N samples (N-SH2, C-SH2 and tSH2) was achieved by growing the bacteria in minimal medium con- taining 15NH4Cl (1 g/l, Cambridge Isotope Laboratories) and 13C-D-glucose (4 g/l, Cambridge Isotope Laboratories) as the sole nitrogen and carbon sources, respectively. Due to its larger size and tendency to form oligomers, tSH2 in complex with the bidentate ITIM-[dPEG4]2-ITSM peptide required sparse deuteration, which was achieved by grow- ing the cells in deuterated minimal medium with protonated carbon source.
After protein expression, the cultures were harvested, pel- leted and stored at − 20 °C until further use. Cells were lysed by sonication in wash buffer (1 M NaCl, 50 mM Tris–HCl, 5% glycerol, 10 mM imidazole, 5 mM β-mercaptoethanol, pH 7.6) supplemented with one tablet of EDTA-free cOm- plete™ protease inhibitors (Roche), 100 µg of lysozyme (Roth) and 50 µg of DNAse (NEB). The lysate was clarified
by centrifugation at 18000×g for 1 h and the filtered super- natant was loaded on a HisTrap HP column (GE Healthcare), previously equilibrated with wash buffer. After loading, the column was washed extensively (10 column volumes, CV) with wash buffer, before elution of the bound protein with 5 CV of elution buffer (1 M NaCl, 50 mM Tris–HCl, 5% glyc- erol, 500 mM imidazole, 5 mM β-mercaptoethanol, pH 7.6).
The fractions containing the protein were pooled and 3C protease (1:100 protease:protein ratio) was added to cleave the His6-thioredoxin tag. Excess imidazole was removed by dialyzing the eluate against 2 l of wash buffer at 4 °C over- night. Purification continued the following day with a second round of affinity chromatography (HisTrap) to separate the His6-thioredoxin tag from the target protein. The fractions containing the protein of interest were concentrated to a final volume of 1−2 ml and loaded on a HiLoad 16/600 Superdex 75 pg column (GE Healthcare), previously equilibrated with NMR buffer (100 mM MES, 150 mM NaCl, 3 mM TCEP, 0.05% NaN3, pH 6.8). The fractions containing pure protein were pooled, the protein concentrated to the desired con- centration and either used directly for experiments or flash- frozen with liquid nitrogen for long-term storage at − 80 °C.
Sample purity was confirmed by SDS-PAGE.
PD-1 immune tyrosine motifs were purchased as syn- thetic phosphopeptides (ITIM: Ac-FSVDpYGELDFQ- NH2; ITSM: Ac-EQTEpYATIVFP-NH2) from Caslo ApS (Lyngby, Denmark). The bidentate peptide ITIM-[dPEG4]2- ITSM was made by connecting ITIM and ITSM with two discrete poly-(ethylene glycol)-4 units, in order to match the length of the linker that separates ITIM and ITSM in wild-type PD-1, and was purchased from the same com- pany (Sequence: Ac-FSVDpYGELDFQ-[dPEG4]-[dPEG4]- EQTEpYATIVFP-NH2).
NMR spectroscopy
NMR assignment spectra were recorded at a temperature of 298 K on Bruker Avance III HD 600 MHz and 850 MHz spectrometers running Topspin 3.2 software and equipped with N2-cooled and He-cooled inverse HCN triple-resonance cryogenic probeheads, respectively. Protein concentrations ranged from 500 µM to 800 µM; for peptide-containing sam- ples, peptides were added in two-fold excess unless specified otherwise below. For tSH2 in complex with ITIM-[dPEG4]2- ITSM, the peptide was in 1.5-fold excess with respect to the protein.
Backbone resonances of N-SH2, C-SH2 and their ITIM- and ITSM-bound forms, and those of unbound tSH2, were assigned using the standard suite of triple-resonance experi- ments (2D 15N-HSQC, 3D HNCO, 3D HNCACB and 3D HN(CO)CACB) (Kay et al. 1994; Muhandiram and Kay 1994; Yamazaki et al. 1994) and the sidechain resonances of N-SH2–ITIM were assigned from 3D HC(C)H-TOCSY
(Kay et al. 1993), 3D H(CCCO)NH-TOCSY (Logan et al.
1992), 3D NOESY-15N-HSQC (Marion et al. 1989), 2D HBCB(CGCD)HD, 2D HBCB(CGCDCE)HE (Yamazaki et al. 1993) and 2D constant-time 13C-HSQC spectra (Vuis- ter and Bax 1992). Assignments of proton resonances of ITIM in complex with N-SH2 were obtained from 2D
13C,15N-filtered NOESY and 2D 13C,15N-filtered TOCSY spectra (Zwahlen et al. 1997) recorded on a sample in which the protein was in excess with respect to the peptide;
deuterated PIPES instead of unlabeled MES was used as a buffer in order to minimize the t1-noise from buffer peaks.
For tSH2 in complex with ITIM-[dPEG4]2-ITSM, backbone resonance assignment was achieved by TROSY-based triple- resonance experiments (2D TROSY-HSQC, 3D TROSY- HNCO, 3D TROSY-HNCACB and 3D TROSY-HN(CO) CACB) (Pervushin et al. 1997; Salzmann et al. 1998) and by chemical-shift comparison with previously assigned N-SH2 and C-SH2 complexes. All the spectra were processed with Topspin 3.2 (Bruker) or NMRpipe (Delaglio et al. 1995).
Peak picking and resonance assignment were done with Ccp- Nmr Analysis (Vranken et al. 2005).
Assignments and data deposition
The features of the 15N HSQC spectra of N-SH2 and C-SH2 confirmed that these two domains adopt a stable fold in solution (Fig. 1). Excluding prolines and the N-terminal residues generated by 3C protease cleavage (whose signals were missing in the spectra, probably due to rapid solvent exchange), the backbone assignments of N-SH2 and C-SH2 were 93% and 96.4% complete, respectively. For N-SH2, the missing signals belonged to the segment R32–G39 (BC loop), which is part of the phosphotyrosine binding region.
This segment is known to be poorly structured in SH2 domains in the absence of bound phosphopeptides (Booker et al. 1992). Similarly, the amide peaks of Q140 and S141 of C-SH2 were missing. Furthermore, the amide peaks of residues H85 and G86 of N-SH2, which join the BG loop to αB, were also missing.
As expected, addition of PD-1-derived phosphopep- tides led to the structuring of the pY-binding region: the assignments of N-SH2–ITIM and N-SH2–ITSM were complete except for G86 and N92, while the assignment of C-SH2–ITSM was 100% complete. On the other hand, in the C-SH2–ITIM complex several amide peaks were missing (G115, G154, N161, V181, G182 and L206) (Figs. 2 and 3). In general, the chemical shifts perturbations induced by ITIM and ITSM on the SH2 domains of SHP2 are different.
The 15N HSQC spectrum of unbound tSH2 closely resembles the overlay of the spectra of unbound N-SH2 and C-SH2, which made the assignment relatively simple by comparison of the chemical shifts. The assignments could be transferred from the isolated SH2 domains to
105A 47R
8H 57Q
102L 43L
55K 96I
54I 46R 53H
48N 103N
98L 2A
52T
97E 7F
44S 88L
89K 72A 45V 26D 56I58N
91K 50A 77L
28S 19L 74L
69E 94D 31A
95V 104C 16A 4R
15E 64D 99K
14V 90E51V 82M
5R
40D 23R
75A 66Y
65L 78V
61D 70K
63Y 20L 80Y
84H 71F 41F
42T
79Q
81Y 76E
32R 59T
21L 17E 100Y
29F
18N92N 87Q 83E
11I
30L 12T 62Y
10N 60G 3S 27G
39G
6W 25V
13G
93G 22T
68G 67G 24G
49G
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0 10.5
105
110
115
120
125
130
73T
149L 152R
173R
220R
175Q 210L
170V
171M 117L
114H
169H204E 116H
216L 176E 150S 172I 105A
217N 174C 212L
168T 113F
138R 118S 203V
166K 151V 209V
124K 211Q 193L
190L 143H
153T
110E 122A
213K 106D
132H 202M
139E 167V
192D
188D 134S 120K
185E 156D
159E 155D 148V
206L 111R 104M 157K 164K 179Y
137V
121E 187F 194V
128E 186R 180D
162D
198K 125L 146D
196H 200N
147F 197Y178K
165S
109S 135F 123E131K
133G 126L
199K 160S
195E 140S 208T 214Q 127T 219T 145G
136L 191T
218T 129K 182G 181V
119G 112W
177L 158G163G 189S 184G 207G 183G
130G
205T
108T
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0 10.5
110
115
120
125
130 A
B
δ( 15N) [ppm]
δ(1H) [ppm]
δ( 15N) [ppm]
δ(1H) [ppm]
Fig. 1 2D 1H,15N HSQC spectra of N-SH2 (a) and C-SH2 (b) with the corresponding assignments. The spectra were collected on a 600 MHz Bruker Avance III spectrometer
36S 39G 41F 32R71F
2A 5R
6W
7F
8H
10N 12T 11I
13G
14V 15E
16A
17E 18N
19L 20L 21L
22T
23R
24G
25V
26D 27G
28S
29F 30L
31A
34S
35K
37N
85H69E 40D 42T
43L 44S
45V
46R
47R 48N 49G
50A 51V
52T
53H 54I
55K 56I
57Q 58N
59T 60G
61D 62Y
63Y
64D
65L 66Y
67G
68G
70K
72A
73T
74L 75A
76E
77L 78V
79Q80Y 81Y
82M
83E
84H 87Q
88L
89K 90E 91K
93G
94D 95V
96I 97E 98L
99K 100Y
102L 103N 104C
105A
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0 10.5
105
110
115
120
125
130
149L 210L 117L
152R
141Q 171M 175Q 220R
116H173R 169H 114H 172I
209V 216L 150S
105A 204E
217N 170V176E
174C 168T 212L 143H
138R 118S 166K 113F
213K 151V
124K 153T 193L
190L 167V 122A
211Q 202M 110E
203V120K106D 132H 192D
185E 198K121E
156D 155D
159E 188D
134S
164K 139E 104M 157K
194V 137V 111R
187F 179Y180D 128E 186R
162D 125L
196H 148V
200N
147F 197Y 178K
142S 205T123E 165S 109S 135F 199K
126L
208T 145G 133G131K 160S 195E127T 140S
136L 219T 191T 214Q
218T 129K
119G 177L 158G 108T 112W
163G 189S
184G 207G 183G 130G
146D
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0 10.5
110
115
120
125
130 A
B
δ( 15N) [ppm]
δ(1H) [ppm]
δ(15N) [ppm]
δ(1H) [ppm]
Fig. 2 2D 1H,15N HSQC spectra of N-SH2–ITIM (a) and C-SH2–ITIM (b) with the corresponding assignments. The spectra were collected on a 600 MHz Bruker Avance III spectrometer
105A
35K 47R 8H
57Q 102L
55K 96I
46R
43L 98L 103N97E
48N 2A
52T 91K53H 89K 7F
54I 28S72A 45V 26D 94D58N 50A74L 77L
85H 44S 69E
31A 104C 19L 37N
56I16A 90E 4R
64D 88L
14V 15E 99K
51V 82M40D
5R
23R 95V
78V 75A
70K
20L 80Y
84H
63Y 71F 65L
42T 32R 79Q
81Y 21L 61D
41F
17E29F 76E
100Y 36S
18N 66Y 87Q
30L 39G 83E 62Y
59T 3S 27G 10N
11I 12T 60G
6W
13G 25V 93G 22T
68G 24G
49G
67G 73T
34S
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0 10.5
105
110
115
120
125
130
177L 158G 163G 184G
207G
104M
105A 106D
108T
109S
110E 111R
112W
113F
114H 116H
117L 118S
119G
120K 122A 121E
123E
124K 125L 126L
127T
128E 129K
130G
131K
132H
133G
134S
135F 136L
137V
138R
139E
140S
141Q 142S
143H 145G
146D 148V 147F
149L 150S
151V
152R 153T
155D
156D 157K 159E
160S
161N 162D
164K 165S
166K 167V
168T 174C 169H 212L 170V
171M 172I
175Q173R 176E
178K 179Y 180D
181V 182G
183G
185E
186R 188D 187F
189S
190L
191T
192D
193L 194V
195E
196H 197Y
198K
200N 199K
202M
203V 204E
205T
206L 208T
209V
210L
211Q
213K
214Q
216L 217N 218T
219T
220R
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0 10.5
110
115
120
125
130
A
B
δ(
15N) [ppm]
δ(
1H) [ppm]
δ(
15N) [ppm]
δ(
1H) [ppm]
Fig. 3 2D1H,15N HSQC spectra of N-SH2–ITSM (a) and C-SH2–ITSM (b) with the corresponding assignments. The spectra were collected on a 600 MHz Bruker Avance III spectrometer
47R
149L 105A
57Q 8H
152R 102L
43L
173R 55K 175Q
210L 220R
170V 171M 96I
54I 46R 117L
169H 114H 116H 216L103N 53H204E 48N
176E 150S 172I 217N2A
174C98L 212L 52T
88L
168T 97E 7F113F
44S 118S
89K
72A 45V
56I 203V
166K 209V
91K26D
151V 50A124K
28S 193L 77L
19L 211Q
190L104C 69E 110E
213K
94D 153T
122A
31A 95V
106D
16A 4R 143H
132H 139E 202M
167V
192D
188D 134S 120K 185E
15E 156D
155D 159E 99K 148V 1M
164K14V
64D 111R 157K
137V
90E 194V
121E
51V 186R
187F
40D 180D 23R
128E 162D
198K 75A 125L
66Y
146D
65L 78V 61D
63Y
20L 196H
80Y 178K
71F 41F 42T 147F
79Q 76E197Y 165S
32R 123E 17E 109S 100Y 135F
126L 18N
133G 160S219T 205T127T 92N 208T 214Q 136L 145G
30L 12T 60G191T 62Y
10N
218T129K 142S 3S 181V
182G 27G 39G
6W 119G
25V
158G 112W
177L 163G93G 22T 189S 184G
68G 207G
183G 67G 130G 24G
49G
154G
11I
199K 108T
140S
29F
74L 5R 131K
21L 59T
138R 58N
141Q
161N 206L
70K 195E
13G
87Q
179Y 200N
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0
10.5
105 110 115 120 125 130
73T
210L 117L
47R 8H
149L
57Q 141Q
171M 152R 102L
96I 220R 46R 55K
55K*
172I 116H175Q 96I*
209V 43L 173R
114H 169H 103N
150S 98L 216L217N 53H*2A 176E
212L
170V 48N
174C 53H
52T 97E* 7F
52T* 89K
54I 138R 45V
45V* 166K 113F
213K 118S26D 50A
58N
72A 58N*
151V 77L
94D 124K 193L
190L 167V
202M 104C185E 74L
44S 153T122A
110E
69E 198K
31A 19L 204E 211Q
4R
56I 16A 120K
88L 132H
155D
192D 156D
134S 15E 159E
121E 164K 14V 1M
194V
188D 187F 139E 157K
137V 111R
82M
51V208T 40D
179Y 128E
186R
180D 23R 146D
125L 75A 5R
196H70K
20L 80Y 65L
178K 84H 147F
63Y 148V
197Y 71F
200N
123E 165S
32R
79Q
61D 199K
41F 21L 17E 135F
17E*
29F 76E 100Y*
126L 195E
87Q 133G
18N 18N*
145G 66Y
39G 131K
160S 127T
136L 219T 140S
30L*
83E83E*
191T 214Q
205T 3S 62Y
129K
11I 27G* 10N*
27G 12T 60G
154G
119G 182G
25V 181V
13G 177L 112W
158G
22T 189S 93G
163G184G207G 183G 130G
24G 68G
49G
67G*
73T
42T
206L
64D 203V
168T
162D 10N
218T
59T
28S
6W
6.5 7.0
7.5 8.0
8.5 9.0
9.5 10.0
10.5
105 110 115 120 125 130
68G*
93G*
30L
54I*
41F*
13G*
91K*91K
57Q*
A
B
97E 89K*
67G
102L*
δ(15N) [ppm]
δ(1H) [ppm]
δ(15N) [ppm]
δ(1H) [ppm]
Fig. 4 2D 1H,15N HSQC spectrum of tSH2 (a) and 2D 1H,15N TROSY HSQC spectrum of tSH2–ITIM-[dPEG4]2-ITSM (b) with the corresponding assignments. In the latter, the resonances belong-
ing to the state in which N-SH2 is bound to ITIM are marked with an asterisk. The spectra were collected on a 850 MHz Bruker Avance III spectrometer
the tSH2 construct, except for the BC loop of N–SH2 (S34–N37) and the Y81–H85 segment (Fig. 4a). The bound state of tSH2 was measured in the presence of a 1.5-fold excess of biphosphorylated ITIM-[dPEG4]2-ITSM
and a tSH2 concentration of 500 µM; the 15N TROSY spectrum at this stoichiometric ratio and protein con- centration reveals the presence of two distinct peaks for several amide groups (Fig. 4b). By comparison of this
Fig. 5 Excerpt of the 13C,15N- filtered TOCSY spectrum of N-SH2–ITIM, which shows the assignment of the amide region of ITIM. H* indicates the N-terminal amide hydro- gen. The spectra were collected on a 600 MHz Bruker Avance III spectrometer
(-4)PheH*,Ha (-3)SerH,Ha
(+6)GlnH,Ha
(-2)ValH,Ha (-3)SerH,Hba (-4)PheH*,Hbb (-4)PheH*,Hba
(-4)PheHd*,Hbb (-1)AspH,Hbb
(-1)AspH,Hba (+4)AspH,Hbb (+6)GlnH,Hga
(+2)GluH,Hgb
(+2)GluH,Hga (+6)GlnH,Hba (+2)GluH,Hbb
(+2)GluH,Hba (-2)ValH,Hb
(-2)ValH,Hgb*
(-2)ValH,Hga*
(+4)AspH,Hba (+3)LeuH,Hdb*
(-4)PheHd*,Hba
(+6)GlnHe2b,He2a
(0)PtrHd1,Hd1 (0)PtrHd1,He1
(0)PtrHe1,He1 (+5)PheHd*,Hd*
(-4)PheHe*,He*
(-4)PheHe*,Hd*
(+5)PheHd*,He*
(+5)PheHe*,He*
(-4)PheHd*,Hd*
(+6)GlnHe2b,He2b
(-2)ValH,H
(-4)PheH*,H*
(-3)SerH,H
(+4)AspH,H (+6)GlnH,H (0)PtrH,H
(+3)LeuH,H (-1)AspH,H (0)PtrH,Hba
(+5)PheH,Hba
(+5)PheH,Ha
(+5)PheH,H (+2)GluH,H
(+1)GlyH,H
(+6)GlnHe2a,He2a
6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
δ(
1H) [ppm]
δ(
1H) [ppm]
spectrum with those of N-SH2–ITIM, N-SH2–ITSM, C-SH2–ITIM and C-SH2–ITSM, it was possible to estab- lish that the two resonances represent two different states of tSH2, one in which N-SH2 is bound to ITIM and one in which N-SH2 is bound to ITSM. The C-SH2 appears to be bound only to ITSM. This indicates that binding of ITIM-[dPEG4]2-ITSM to tSH2 results in a heterogeneous mixture of complexes of different architecture. Contrary to the bound states of isolated N-SH2, the peaks of the BC loop were still missing in this complex. In addition, the segment A105–T108, corresponding to the linker between N-SH2 and C-SH2, did not yield any observable amide peaks, presumably due to either an unfavorable confor- mational exchange regime or rapid proton-exchange with the solvent. Due to its physiological importance (ITIM is the specific binder of N-SH2 and is directly responsible for maximum activation of SHP2), the assignment of the resonances of the N-SH2-ITIM complex also included pro- tein side-chains and peptide proton resonances (Fig. 5).
The 1H, 13C, 15N backbone (1HN, 15N, 13Cα, 13Cβ,
13CO) resonance assignments of N-SH2, C-SH2, tSH2, C-SH2–ITIM and N-SH2–ITSM have been deposited at the BioMagResBank (https ://www.bmrb.wisc.edu) under accession codes 28069, 28070, 28071, 28072 and 28073, respectively. The backbone and sidechain resonances of N-SH2–ITIM are available under accession code 28074, while those of C-SH2–ITSM have been published as part of previous work (BMRB code 34384) (Marasco et al.
2020). Due to the heterogeneous population of bound states, the backbone chemical shifts of the tSH2–ITIM- [dPEG4]2-ITSM have been deposited as two separate groups: those corresponding to the state in which N-SH2 is bound to ITIM (BMRB code 28075) and those corre- sponding to the state in which N-SH2 is bound to ITSM (BMRB 28076).
Acknowledgements Open Access funding provided by Projekt DEAL.
This work was funded by the German Science Foundation DFG (Grant CA 294/20-1). MM was supported by a fellowship from the Hannover School for Biomolecular Drug Research (HSBDR) and was a member of the Hannover Biomedical Research School (HBRS) and the MD/
PhD program “Molecular Medicine”.
Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
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