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PreprintCited by 0T-cell and B-cell ImmunologyProtein Tyrosine PhosphatasesProtein Kinase Regulation and GTPase Signaling

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Themis and Grb2 form a constitutive structural hub in T cell receptor signalling

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Savvides, Savvas, Clancy, Danielle, Sanjuan, Alba Sanz, Gillis, Elisabeth, Velghe, Imke, Droogenbroeck, Yana Van, Felix, Jan, Bloch, Yehudi, Cuadrado, Alvaro Furones, Merceron, Romain, Schenck, Stephan, Vandenabeele, Peter, Brunner, Janine, TAGHON, TOM, Elewaut, Dirk, Tougaard, Peter·2025·Nature Communications
DOI 10.21203/rs.3.rs-6042814/v1PMC PMC13381594

Preprint — published as 10.1038/s41467-026-73359-8 · Paper page

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Abstract

Positive selection of thymocytes is essential for laying the foundations of the mammalian immune system that include the T cell repertoire, self-tolerance, and prevention of autoimmunity. Themis, the archetypal member of a metazoan protein family featuring distinctive CABIT domains, crucially regulates thymocyte positive selection by linking signalling by the T cell receptor (TCR) to the linker of activation of TCR (LAT). Intriguingly, Themis has been proposed to function via a constitutive complex with the multifunctional adaptor Grb2. Although poised to represent a paradigm shift in our understanding of TCR signalling, the structural and mechanistic basis of such an assembly has remained enigmatic. Here, we present the cryo-EM structure of Themis in complex with Grb2, which reveals how the tandem CABIT domains of Themis engulf the C-terminal SH3 domain of Grb2 (Grb2 SH3C ) to enable its latching onto the proline rich sequence of Themis. The remaining two domains of Grb2 adopt at least three conformational poses set to interact with other binding partners such as Sos1. Structural insights from unbound Themis unmask the pronounced flexibility of the CABIT domains of Themis, which becomes ordered upon binding to Grb2 to create a binding hotspot for their constitutive complex. Indeed, Themis variants that abrogate interactions with Grb2 also fail to activate the tyrosine phosphatase SHP-1 after TCR stimulation, analogous to the functional phenotype of Themis-deficient cells. Collectively, our study draws the blueprint of the Themis-Grb2 complex as a dynamic structural hub in T cell development.

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Fig. 1 Biochemical and biophysical characterisation of the Themis1–563-Grb2 complex. a Schematic domain delineations, representative chromatograms and SDS-PAGE gels for the purification of Themis1-–563 (top) and Grb2FL (bottom). Chromatograms and SDS-PAGE analysis are representative of different protein batches of at least five independent experiments each. b Representative isothermal titration calorimetry (ITC) thermograms for the titration of Themis1–563 (7 μM) with Grb2FL (41.6 μM). DP, differential electrical power. Fitted values are provided with their fitting errors as reported by the MicroCal PEAQ-ITC Analysis software version 1.1.0.1262. Similar results were obtained from four independent experiments. c Western blot analysis of SHP-1 phosphorylation (pY564) after TCR stimulation. Jurkat cells reconstituted with an empty lentiviral vector (control), Themis-deficient Jurkat cells (KO) generated by CRISPR/Cas9 gene editing and Jurkat KO cells reconstituted with Themis1–563 were stimulated with plate-bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL) for the indicated time period. The indicated proteins were analyzed by Western blot. Data are representative of three independent experiments. d Representative chromatogram for the co-expression and purification of the Themis1–563-Grb2FL complex from HEK293 suspension cells. Data are representative of at least six independent experiments.
Fig. 1 Biochemical and biophysical characterisation of the Themis1–563-Grb2 complex. a Schematic domain delineations, representative chromatograms and SDS-PAGE gels for the purification of Themis1-–563 (top) and Grb2FL (bottom). Chromatograms and SDS-PAGE analysis are representative of different protein batches of at least five independent experiments each. b Representative isothermal titration calorimetry (ITC) thermograms for the titration of Themis1–563 (7 μM) with Grb2FL (41.6 μM). DP, differential electrical power. Fitted values are provided with their fitting errors as reported by the MicroCal PEAQ-ITC Analysis software version 1.1.0.1262. Similar results were obtained from four independent experiments. c Western blot analysis of SHP-1 phosphorylation (pY564) after TCR stimulation. Jurkat cells reconstituted with an empty lentiviral vector (control), Themis-deficient Jurkat cells (KO) generated by CRISPR/Cas9 gene editing and Jurkat KO cells reconstituted with Themis1–563 were stimulated with plate-bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL) for the indicated time period. The indicated proteins were analyzed by Western blot. Data are representative of three independent experiments. d Representative chromatogram for the co-expression and purification of the Themis1–563-Grb2FL complex from HEK293 suspension cells. Data are representative of at least six independent experiments.CC BY-NC-ND · PubMed Central
Fig. 2 Structure of the Themis-Grb2 complex. a Schematic representation of the domain organisation of Themis1–563 and Grb2 and sample preparation of the Themis1–563-Grb2-PMb256 complex for cryo-EM. Representative SEC chromatogram and SDS-PAGE analysis of the Themis1–563-Grb2 complex incubated with a 2 molar excess of PMb256. The second SEC peak corresponds to excess PMb256. The data are representative of two independent experiments. Newly delineated domain boundaries based on structural models are indicated. b Representative cryo-EM 2D class averages of the Themis1–563-Grb2-PMb256 complex. Scale bars represent 100 Å. c Cryo-EM map of the Themis1–563-Grb2-PMb256 complex. The map was sharpened using DeepEMhancer, contoured at 0.12 and coloured per domain with ThemisCABIT1 domain in cyan, ThemisCABIT2 in dark blue, ThemisPRS in orange, Grb2SH3C in pink and PMb256 in grey. d Real-space refined atomic model of the Themis1–563-Grb2SH3C complex. Cartoon representations of front and back views are shown, with Grb2SH3C depicted in surface view and the PMb256 model removed for clarity. Side view of the complex shown with a surface representation of Themis, illustrating how Grb2SH3C is engulfed by the CABIT domains. The model is coloured per domain, as in (c). e Zoom-in views of the three interaction sites in the Themis1–563-Grb2SH3C interface fitted in a local refinement 3D map displayed as a grey mesh. Grb2SH3C, ThemisPRS, ThemisCABIT1, ThemisCABIT2 are shown in pink, orange, cyan and dark blue, respectively.
Fig. 2 Structure of the Themis-Grb2 complex. a Schematic representation of the domain organisation of Themis1–563 and Grb2 and sample preparation of the Themis1–563-Grb2-PMb256 complex for cryo-EM. Representative SEC chromatogram and SDS-PAGE analysis of the Themis1–563-Grb2 complex incubated with a 2 molar excess of PMb256. The second SEC peak corresponds to excess PMb256. The data are representative of two independent experiments. Newly delineated domain boundaries based on structural models are indicated. b Representative cryo-EM 2D class averages of the Themis1–563-Grb2-PMb256 complex. Scale bars represent 100 Å. c Cryo-EM map of the Themis1–563-Grb2-PMb256 complex. The map was sharpened using DeepEMhancer, contoured at 0.12 and coloured per domain with ThemisCABIT1 domain in cyan, ThemisCABIT2 in dark blue, ThemisPRS in orange, Grb2SH3C in pink and PMb256 in grey. d Real-space refined atomic model of the Themis1–563-Grb2SH3C complex. Cartoon representations of front and back views are shown, with Grb2SH3C depicted in surface view and the PMb256 model removed for clarity. Side view of the complex shown with a surface representation of Themis, illustrating how Grb2SH3C is engulfed by the CABIT domains. The model is coloured per domain, as in (c). e Zoom-in views of the three interaction sites in the Themis1–563-Grb2SH3C interface fitted in a local refinement 3D map displayed as a grey mesh. Grb2SH3C, ThemisPRS, ThemisCABIT1, ThemisCABIT2 are shown in pink, orange, cyan and dark blue, respectively.CC BY-NC-ND · PubMed Central
Fig. 3 Structural and functional interrogation of Themis1–563 mutants. a Experimental BLI setup and representative response curves fitted with a 1:1 binding model (red) to quantify the kinetics (ka, kd) and binding affinity (KD) of wild-type and mutant Themis1–563 to Grb2FL. The location of the mutations in the Themis1–563-Grb2SH3C interface is indicated on a surface representation of the complex. b Sequence alignment of ThemisPRS with proline-rich motifs from other Grb2SH3C binding partners, Gab1, Gab2 and SLP-76. c Representative BLI response curves to quantify the interaction of Grb2FL and Grb2SH3C to an immobilised biotinylated ThemisPRS peptide. The peptide sequence is shown in an inset. d Zoom-in view of the ThemisCABIT2 domain showing residue L411 coloured in red. e SEC chromatogram and SDS-PAGE analysis for the purification of the ThemisL411V mutant variant. The data are representative of two independent experiments. f Representative BLI response curves fitted with a 1:1 binding model (red) to compare the kinetics (ka, kd) and binding affinity (KD) of ThemisWT and ThemisL411V to immobilised Grb2. For all BLI experiments, start and end concentrations of the two-fold dilution series are shown as an inset.
Fig. 3 Structural and functional interrogation of Themis1–563 mutants. a Experimental BLI setup and representative response curves fitted with a 1:1 binding model (red) to quantify the kinetics (ka, kd) and binding affinity (KD) of wild-type and mutant Themis1–563 to Grb2FL. The location of the mutations in the Themis1–563-Grb2SH3C interface is indicated on a surface representation of the complex. b Sequence alignment of ThemisPRS with proline-rich motifs from other Grb2SH3C binding partners, Gab1, Gab2 and SLP-76. c Representative BLI response curves to quantify the interaction of Grb2FL and Grb2SH3C to an immobilised biotinylated ThemisPRS peptide. The peptide sequence is shown in an inset. d Zoom-in view of the ThemisCABIT2 domain showing residue L411 coloured in red. e SEC chromatogram and SDS-PAGE analysis for the purification of the ThemisL411V mutant variant. The data are representative of two independent experiments. f Representative BLI response curves fitted with a 1:1 binding model (red) to compare the kinetics (ka, kd) and binding affinity (KD) of ThemisWT and ThemisL411V to immobilised Grb2. For all BLI experiments, start and end concentrations of the two-fold dilution series are shown as an inset.CC BY-NC-ND · PubMed Central
Fig. 4 Themis CABIT domains are highly flexible and are stabilised by Grb2 binding. a Sample preparation of the Themis1–563-PMb256 complex for cryo-EM. Representative SEC chromatogram and SDS-PAGE analysis of Themis1–563 incubated with a 2 molar excess of PMb256. The second SEC peak contains excess PMb256. Data are representative of one independent experiment. b Representative cryo-EM 2D class averages of the Themis1–563-PMb256 complex. Scale bar represents 100 Å. c Cryo-EM map of the Themis1–563-PMb256 complex. The displayed 3D map is contoured at 0.12 and coloured with Themis CABIT domains in blue, and PMb256 in grey. Gold-standard Fourier Shell Correlation (FSC) curves are shown, with the estimated resolution at FSC = 0.143 (dark blue line) shown for the corrected FSC curve (purple line). d 3D cryo-EM map shown in c coloured according to local resolution. e 3D classification without alignment of the particles contributing to the map shown in (c) results in two classes in which the CABIT1 domain is present (class 1) or absent (class 2). Sharpened maps from subsequent non-uniform refinement are shown contoured at 0.12. f 3D variability analysis of particles from class 2 shows movement of the unbound ThemisCABIT1 domain. 3D volumes from the start (Frame 0, blue) and end (Frame 19, red) of the component analysis are shown.
Fig. 4 Themis CABIT domains are highly flexible and are stabilised by Grb2 binding. a Sample preparation of the Themis1–563-PMb256 complex for cryo-EM. Representative SEC chromatogram and SDS-PAGE analysis of Themis1–563 incubated with a 2 molar excess of PMb256. The second SEC peak contains excess PMb256. Data are representative of one independent experiment. b Representative cryo-EM 2D class averages of the Themis1–563-PMb256 complex. Scale bar represents 100 Å. c Cryo-EM map of the Themis1–563-PMb256 complex. The displayed 3D map is contoured at 0.12 and coloured with Themis CABIT domains in blue, and PMb256 in grey. Gold-standard Fourier Shell Correlation (FSC) curves are shown, with the estimated resolution at FSC = 0.143 (dark blue line) shown for the corrected FSC curve (purple line). d 3D cryo-EM map shown in c coloured according to local resolution. e 3D classification without alignment of the particles contributing to the map shown in (c) results in two classes in which the CABIT1 domain is present (class 1) or absent (class 2). Sharpened maps from subsequent non-uniform refinement are shown contoured at 0.12. f 3D variability analysis of particles from class 2 shows movement of the unbound ThemisCABIT1 domain. 3D volumes from the start (Frame 0, blue) and end (Frame 19, red) of the component analysis are shown.CC BY-NC-ND · PubMed Central
Fig. 5 Grb2 SH3N and SH2 domains are highly flexible and sample multiple conformational states. a The particle stack contributing to the consensus map (Fig. 2d) was further processed using 3D classification without alignment in cryoSPARC using 10 classes as input. The resulting classes are shown, with four classes showing volume corresponding to Grb2SH3N and Grb2SH2 (dashed boxes). b Maps corresponding to class 1–3 from 3D classification were further processed by particle subtraction to remove the PMb moiety, followed by local refinement. The displayed 3D maps are contoured at 0.07 and coloured as before, with the additional Grb2SH3N and Grb2SH2 domains in dark pink and wine red, respectively. Particle numbers (p.) contributing to the map for each class are shown. For all maps, the back view of the complex is shown. c Schematic showing the distinct conformational states of Grb2 captured by cryo-EM analysis. Grb2SH3C domain is locked in one position through its interaction with Themis, while Grb2SH2 and Grb2SH3N exhibit 180-degree movement at the back of the Themis-Grb2 molecule. Surface representations of Grb2SH2 and Grb2SH3N are shown based on rigid body fitting of individual domains in the cryo-EM maps from (b) in ChimeraX.
Fig. 5 Grb2 SH3N and SH2 domains are highly flexible and sample multiple conformational states. a The particle stack contributing to the consensus map (Fig. 2d) was further processed using 3D classification without alignment in cryoSPARC using 10 classes as input. The resulting classes are shown, with four classes showing volume corresponding to Grb2SH3N and Grb2SH2 (dashed boxes). b Maps corresponding to class 1–3 from 3D classification were further processed by particle subtraction to remove the PMb moiety, followed by local refinement. The displayed 3D maps are contoured at 0.07 and coloured as before, with the additional Grb2SH3N and Grb2SH2 domains in dark pink and wine red, respectively. Particle numbers (p.) contributing to the map for each class are shown. For all maps, the back view of the complex is shown. c Schematic showing the distinct conformational states of Grb2 captured by cryo-EM analysis. Grb2SH3C domain is locked in one position through its interaction with Themis, while Grb2SH2 and Grb2SH3N exhibit 180-degree movement at the back of the Themis-Grb2 molecule. Surface representations of Grb2SH2 and Grb2SH3N are shown based on rigid body fitting of individual domains in the cryo-EM maps from (b) in ChimeraX.CC BY-NC-ND · PubMed Central
Fig. 6 Themis-Grb2 complex acts as a signalling hub downstream of TCR activation. a Western blot analysis of SHP-1 phosphorylation (pY564) after TCR activation. Themis-deficient Jurkat cells (KO) were transduced with a lentiviral vector expressing full-length wild-type Themis (WT) or Themis mutants, as indicated. Jurkat cells reconstituted with an empty lentiviral vector were used as a control. Cells were stimulated with plate-bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL). The indicated proteins were analysed by Western blot. Band intensities for p-SHP-1, p-LAT, p-Vav1 and p-PLCγ1 were quantified and normalised to total ERK. Values below the blots indicate quantified fold changes in phosphorylation relative to untreated control cells. Data are representative of three independent experiments. b Sequence alignment of the ThemisPRS and Sos1PRS peptide. Residue numbers for each peptide are indicated. c Representative response curves fitted with a 1:1 binding model (red) to quantify the kinetics (ka, kd) and binding affinity (KD) of Grb2FL, Grb2SH3C and d Themis1–563-Grb2FL complex to Sos1 PRS peptide. Schematics show the BLI setup for each experiment. Start and end concentrations of the two-fold dilution series are shown as an inset. e Schematic depicting the role of the Themis-Grb2 complex as a proximal signalling hub downstream of the TCR. Upon TCR stimulation, the tyrosine kinase Lck is recruited to the membrane via the CD4 and CD8 co-receptors. Lck phosphorylates immunoreceptor tyrosine-based activation motifs (ITAM) in the TCR CD3 chains, facilitating the recruitment of the protein tyrosine kinase ZAP70. ZAP70 phosphorylates the transmembrane adaptor protein LAT, promoting the formation of the LAT signalosome. The Themis-Grb2 complex is recruited to the membrane via Grb2SH2 binding to phosphorylated LAT. Once there, Themis is positioned to interact with the protein tyrosine phosphatase SHP-1 and regulate its activity to fine-tune TCR signalling. Simultaneously, the Grb2SH3N domain is available for binding to other target proteins, such as Sos1, contributing to the regulation of other downstream signalling pathways. Tyrosine phosphorylation (P) is depicted in yellow.
Fig. 6 Themis-Grb2 complex acts as a signalling hub downstream of TCR activation. a Western blot analysis of SHP-1 phosphorylation (pY564) after TCR activation. Themis-deficient Jurkat cells (KO) were transduced with a lentiviral vector expressing full-length wild-type Themis (WT) or Themis mutants, as indicated. Jurkat cells reconstituted with an empty lentiviral vector were used as a control. Cells were stimulated with plate-bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL). The indicated proteins were analysed by Western blot. Band intensities for p-SHP-1, p-LAT, p-Vav1 and p-PLCγ1 were quantified and normalised to total ERK. Values below the blots indicate quantified fold changes in phosphorylation relative to untreated control cells. Data are representative of three independent experiments. b Sequence alignment of the ThemisPRS and Sos1PRS peptide. Residue numbers for each peptide are indicated. c Representative response curves fitted with a 1:1 binding model (red) to quantify the kinetics (ka, kd) and binding affinity (KD) of Grb2FL, Grb2SH3C and d Themis1–563-Grb2FL complex to Sos1 PRS peptide. Schematics show the BLI setup for each experiment. Start and end concentrations of the two-fold dilution series are shown as an inset. e Schematic depicting the role of the Themis-Grb2 complex as a proximal signalling hub downstream of the TCR. Upon TCR stimulation, the tyrosine kinase Lck is recruited to the membrane via the CD4 and CD8 co-receptors. Lck phosphorylates immunoreceptor tyrosine-based activation motifs (ITAM) in the TCR CD3 chains, facilitating the recruitment of the protein tyrosine kinase ZAP70. ZAP70 phosphorylates the transmembrane adaptor protein LAT, promoting the formation of the LAT signalosome. The Themis-Grb2 complex is recruited to the membrane via Grb2SH2 binding to phosphorylated LAT. Once there, Themis is positioned to interact with the protein tyrosine phosphatase SHP-1 and regulate its activity to fine-tune TCR signalling. Simultaneously, the Grb2SH3N domain is available for binding to other target proteins, such as Sos1, contributing to the regulation of other downstream signalling pathways. Tyrosine phosphorylation (P) is depicted in yellow.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Savvides, S., Clancy, D., Sanjuan, A. S., Gillis, E., Velghe, I., Droogenbroeck, Y. V., Felix, J., Bloch, Y., Cuadrado, A. F., Merceron, R., Schenck, S., Vandenabeele, P., Brunner, J., TAGHON, T., Elewaut, D., & Tougaard, P.
Affiliations
  • Ghent University (BE)
  • VIB-UGent Center for Inflammation Research
  • Deutsches Elektronen-Synchrotron DESY (DE)
  • Vrije Universiteit Brussel (BE)
  • Ghent University Hospital (BE)
  • University of Copenhagen (DK)
Corresponding author
  • Savvides, Savvas
  • Clancy, Danielle
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-04-23
Volume
17
Issue
1
ISSN
2041-1723 (ISSN-L)
DOI
10.21203/rs.3.rs-6042814/v1
PMC
PMC13381594
OpenAlex
W7161848002
Cited by
0
References
1
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by-nc-nd)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Fonds Wetenschappelijk Onderzoek (G0H1222N, 1219923N, G049820N)
  • Vlaams Instituut voor Biotechnologie (C0101)
  • Vlaamse regering
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Tyrosine Phosphatases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Keywords
THEMIS, Grb2, T cell receptor signaling, thymic selection, SH3 domain binding, protein-protein interaction, interdomain flexibility, adaptor protein complexes, T cell signaling
MeSH terms
Protein Domains, Animals, Humans, Protein Binding, Receptors, Antigen, T-Cell, T-Lymphocytes, Signal Transduction, src Homology Domains, Cryoelectron Microscopy, Intercellular Signaling Peptides and Proteins, Mice, GRB2 Adaptor Protein, Protein Tyrosine Phosphatase, Non-Receptor Type 6

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