Supplemental data for "HACS1 signaling adaptor protein recognizes a motif in the Paired Immunoglobulin Receptor B cytoplasmic domain"
| dc.date.accessioned | 2020-09-07T00:03:54Z | |
| dc.date.available | 2020-09-07T00:03:54Z | |
| dc.date.issued | 2020 | |
| dc.description | NMR dynamics study — A relaxation study at 700 MHz was performed to determine 15N T1 relaxation times (60, 100, 200, 400, 600, 800, 1000 ms delays), 15N T2 relaxation times (10, 30, 50, 70, 90, 110, 130, 150 ms delays) and heteronuclear 15N{1H}NOEs. Using a freshly prepared and reduced 0.3 mM sample of 15N-labeled in a similar NMR buffer except DTT was replaced with 2.5 mM TCEP. 6xHis-tagged HACS1 SH3 domain, relaxation data were acquired as interleaved pseudo-3D spectra. Peak integrations were made with the nLinLS module of NMRPipe, followed by curve fitting to an exponential decay function with LMQUICK. A correlation time was calculated by a global analysis of the relaxation data. SHP2-PIRB molecular model — The coordinates of the SHP2 SH2-N domain crystal structure (PDB: 4QSY) bound to a GAB1 phosphopeptide was used as a starting point to make a molecular model of the SH2 domain bound to PIRB ITIM3. The GAB1 peptide was extended several amino acids with SWISS-MODEL and then modified to match PIRB with Rosetta Remodel. | en_US |
| dc.description.abstract | HACS1 (hematopoietic adaptor containing SH3 and SAM domains-1) is a signaling protein with two juxtaposed protein-protein interaction domains and an intrinsically unstructured region that spans half the sequence. Here, we describe the interaction between the HACS1 SH3 domain and a sequence near the third immunoreceptor tyrosine-based inhibition motif (ITIM3) of the Paired immunoglobulin receptor B (PIRB). From surface plasmon resonance binding assays using mouse and human PIRB ITIM3 phosphopeptides as ligands, the HACS1 SH3 domain and SHP2 N-terminal SH2 domain demonstrated comparable affinities in the micromolar range. Since the PIRB ITIM3 sequence represents an atypical ligand for an SH3 domain, we determined the NMR structure of the HACS1 SH3 domain and performed a chemical shift mapping study. This study showed that the binding site on the HACS1 SH3 domain for PIRB shares many of the same amino acids found in a canonical binding cleft normally associated with polyproline ligands. Molecular modeling suggests that the respective binding sites in PIRB ITIM3 for the HACS1 SH3 domain and the SHP2 SH2 domain are too close to permit simultaneous binding. As a result, the HACS1-PIRB partnership has the potential to amalgamate signaling pathways that influence both immune and neuronal cell fate. | en_US |
| dc.description.sponsorship | Natural Sciences and Engineering Council of Canada | en_US |
| dc.identifier.citation | Communications Biology (in review) | en_US |
| dc.identifier.uri | http://hdl.handle.net/10315/37813 | |
| dc.language.iso | en | en_US |
| dc.rights | CC0 1.0 Universal | * |
| dc.rights.uri | http://creativecommons.org/publicdomain/zero/1.0/ | * |
| dc.title | Supplemental data for "HACS1 signaling adaptor protein recognizes a motif in the Paired Immunoglobulin Receptor B cytoplasmic domain" | en_US |
| dc.type | Dataset | en_US |
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- HACS1-PIRB-CSPs-Figure-4.xlsx
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- Microsoft Excel file containing NMR Chemical shift perturbation data resulting from the addition of a Protein G B1 domain PIRB ITIM3 fusion protein to the sample of 15N-labeled HACS1 SH3 domain (Figure 4 in the manuscript)
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- SHP2-SH2-PIRB-peptide-model.pdb
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- 146.28 KB
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- Protein Data Bank (PDB) text file describing the SHP2 SH2 domain / PIRB ITIM3 molecular model as presented in Figure 6
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- HACS1-SH3-dynamics.xlsx
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- 12.22 KB
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- Microsoft Excel
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- Microsoft Excel file containing NMR 15N-T1, 15N-T2 and {1H-15N} NOE dynamics data (Supplementary Figure S5)
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