We constructed the phage display vector, DsbFNp3FL as follows. We amplified the 5′ portion of the M13 gene 3 from M13mp18 using a forward primer that introduced an MluI site N-terminal to the mature p3 protein (5′-GCATACGCGTGCTGAAACTGTTGAAAG-3′) and a reverse primer annealing to the downstream of a natural ClaI site in M13 gene 3. After digesting with the two enzymes, we cloned the fragment into a phage-display vector containing the gene for the C-terminal domain of p3, described previously,57 (link) to regenerate the full-length p3 gene. Escherichia coli cells used for phage display harbored a LacI-containing plasmid, pMCSG21 (a generous gift of Dr. Mark Donnelly, Argonne National Laboratory), based on pCDFDuet-1 (Novagen). The Abl1 and Abl2 expression vectors were gifts of Dr. Piers Nash, University of Chicago.58 (link) All other SH2 domain genes were from Open Biosystems (catalog number, OHS4902), and we cloned them into a vector for an N-terminal His10-tag fusion protein,51 (link) as described in Supplementary Methods . We constructed alanine mutants by Kunkel mutagenesis as described previously.19 (link)
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Genes & Molecular Sequences
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Amino Acid Sequence
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SH2 Domain
SH2 Domain
The SH2 (Src Homology 2) domain is a protein module that recognizes and binds to specific phosphorylated tyrosine residues in target proteins.
These interactions play a crucial role in signal transduction pathways, mediating the assembly of multi-protein complexes that facilitate the transmission of signals within cells.
SH2 domains are found in a variety of signaling proteins, such as kinases, phosphatases, and adaptor molecules, and their binding specificity is determined by the amino acid sequence surrounding the phosphotyrosine.
Understanding the structure, function, and binding properties of SH2 domains is essential for elucidating the mechanisms of cellular signaling and developing targeted therapies for diseases associated with dysregulated signal transduction, such as cancer and immune disorders.
Reseachers can maximize their SH2 domain studies by using the PubCompare.ai platform, which helps identify the best protocols and products from the literature, preprints, and patents, and leverages AI-driven comparisons to enhance reproducibility and accuracy.
These interactions play a crucial role in signal transduction pathways, mediating the assembly of multi-protein complexes that facilitate the transmission of signals within cells.
SH2 domains are found in a variety of signaling proteins, such as kinases, phosphatases, and adaptor molecules, and their binding specificity is determined by the amino acid sequence surrounding the phosphotyrosine.
Understanding the structure, function, and binding properties of SH2 domains is essential for elucidating the mechanisms of cellular signaling and developing targeted therapies for diseases associated with dysregulated signal transduction, such as cancer and immune disorders.
Reseachers can maximize their SH2 domain studies by using the PubCompare.ai platform, which helps identify the best protocols and products from the literature, preprints, and patents, and leverages AI-driven comparisons to enhance reproducibility and accuracy.
Most cited protocols related to «SH2 Domain»
Alanine
Cells
Cloning Vectors
Enzymes
Escherichia coli
Genes
Gifts
Mutagenesis
Nonalcoholic Steatohepatitis
nucleoprotein, Measles virus
Oligonucleotide Primers
Phage Display Techniques
Pierson syndrome
Plasmids
Proteins
SH2 Domain
Generation of mice with a conditional STAT3 allele has been described previously (9 (link)). Exons 18–20, which contain the SH2 domain of STAT3, were flanked by two loxP sites. Two Tie2-Cre transgenic mouse strains expressing Cre under the control of the TIE2–kinase promoter/enhancer were generated. One was described previously with expression in hematopoietic and ECs (14 (link), 22 (link)), and here, we describe another with a more endothelial-specific expression. The TIE2e-Cre;STAT3f/d mutant mice were generated through TIE2e-Cre;STAT3+/d mice crossed to STAT3f/f mice. The genotype was determined by PCR as described previously (9 (link)). TIE2e-Cre mice were mated with Z/EG reporter mice, which produce green fluorescence protein (GFP) from a constitutive promoter after removal of LacZ by cre-mediated recombination (23 (link)). Transthyretin (TTR) Cre mice were generated in our laboratory (unpublished data) and the α-MHC-Cre mice were from M. Schneider (Baylor College of Medicine, Houston, TX). All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and under the approval of the Yale Medical School Animal Care and Use Committee.
Alleles
Animals
Animals, Laboratory
Endothelium
Exons
Genotype
Green Fluorescent Proteins
Hematopoietic System
LacZ Genes
Mice, Laboratory
Mice, Transgenic
Pharmaceutical Preparations
Phosphotransferases
Prealbumin
Recombination, Genetic
SH2 Domain
STAT3 protein, human
Strains
Synthetic biological probes, YF-iSH and YF-Tiam1, have been previously described [8] (link), [9] (link). Briefly, YF-iSH consists of YFP, FKBP and an inter SH2 domain (420–615) of p85β (gene accession number: BC006796). YF-Tiam1 has a GEF domain (1012–1592) of Tiam1 (U16296) inserted into a YFP-FKBP backbone vector. These probes were always coexpressed with Lyn-FRB, a membrane targeted FRB, in order to induce iRap-mediated plasma membrane localization. The compound iRap was synthesized and provided by Tom Wandless's lab at Stanford [8] (link), [9] (link). All the other chemical reagents were purchased from the following commercial companies: DMSO, Fibronectin, and fMLP from Sigma-Aldrich, Latrunculin A and Cytochalasin D from Calbiochem, DyeCycle from Invitrogen, and PTX from List Biological Laboratories.
Biopharmaceuticals
Cloning Vectors
Cytochalasin D
FN1 protein, human
Genes
IL1RN protein, human
latrunculin A
Plasma Membrane
SH2 Domain
Sulfoxide, Dimethyl
Tacrolimus Binding Proteins
TIAM1 protein, human
Tissue, Membrane
Vertebral Column
Multiple sequence alignments of ten protein families were chosen from the Pfam database (version 7.3) [29 (link)]. These families are: adenylyl kinase (adkinase) (representing structure PDB ID: 1aky; its ligand or substrate: AP5) [47 (link)], guanine nucleotide exchange factor (gef) (1bkd; H-Ras) [48 (link)], globin (1a6g; HEM) [49 (link)], pdz domain (1be9; C-terminal peptide of protein CRIPT) [50 (link)], ph domain (1mai; I3P) [51 (link)], ptb domain (1shc; PTR) [52 (link)], ras (821p; GTN) [53 (link)], sh2 domain (1a09; ACE) [54 (link)], sh3 domain (1nlo; ACE) [55 (link)] and subtilase (1av7; SBL) [56 (link)]. Most of these alignments contain many sequences. We pruned and clustered the sequences in each alignment according to the length and diversity. Representative sequences were kept and used for tree inference and ancestral sequence reconstruction. This procedure was done in three steps: 1) removing fragments, 2) single-linkage clustering and 3) complete-linkage clustering, as described below.
1. For each family, there is a template sequence with known structure. The sequences, which cover less than 75% of the non-gapped positions in the template sequence with amino acids, were considered to be fragments and discarded.
2. A sequence identity matrix was calculated for the remaining sequences. A single linkage clustering was done to form sequence groups at sequence identity threshold 0.8. For each group, we chose the longest sequence as a representative, discarding other members. This step reduced redundancy in the dataset.
3. An average sequence identity was calculated for the remaining sequences. We used this average identity as a threshold for complete linkage clustering to form new sequence groups. Four groups with the largest sequence numbers were chosen to form our new alignment. Any group with the same number of sequences as the fourth group was also included in the new alignment. The purpose of this step is to keep the major sequence subgroups of a family while leaving out highly divergent sequences that might be deleterious for tree inference.
1. For each family, there is a template sequence with known structure. The sequences, which cover less than 75% of the non-gapped positions in the template sequence with amino acids, were considered to be fragments and discarded.
2. A sequence identity matrix was calculated for the remaining sequences. A single linkage clustering was done to form sequence groups at sequence identity threshold 0.8. For each group, we chose the longest sequence as a representative, discarding other members. This step reduced redundancy in the dataset.
3. An average sequence identity was calculated for the remaining sequences. We used this average identity as a threshold for complete linkage clustering to form new sequence groups. Four groups with the largest sequence numbers were chosen to form our new alignment. Any group with the same number of sequences as the fourth group was also included in the new alignment. The purpose of this step is to keep the major sequence subgroups of a family while leaving out highly divergent sequences that might be deleterious for tree inference.
Amino Acid Sequence
Globin
Ligands
Peptides
Phosphotransferases
Pleckstrin Homology Domains
polypeptide C
Protein C
Proteins
Reconstructive Surgical Procedures
Sequence Alignment
SH2 Domain
SH3 Domain
Trees
The protein-peptide complex structures were solved either by NMR or x-ray crystallography. The interaction of the N-SH2 and C-SH2 domains as well as of SHP21–220 with ITIM, ITSM, and ITIM-[dPEG4]2-ITSM was analyzed by NMR. The multimerization state of the complexes was studied by SEC-MALS. The binding affinities were determined by NMR and calorimetry.
Calorimetry
Crystallography, X-Ray
Peptides
Proteins
SH2 Domain
Most recents protocols related to «SH2 Domain»
SAXS data reduction, buffer subtraction, and further analysis were performed using BioXTAS RAW version 2.1.1107 (link). An average of 30 frames prior to an eluted peak was used for buffer subtraction. Protein peaks were also run through evolving factor analysis (EFA) to deconvolute peaks into the individual scattering components where applicable. The forward scattering intensity I(0) and radius of gyration (Rg) were calculated from the Guinier fit. The normalized Kratky plot, pair distance distribution plot, or P(r), and Porod volume (VP) were calculated using the program GNOM embedded in the BioXTAS RAW software86 (link). The calculation of theoretical scattering curves for the crystal structure PDB 1GRI was performed using the program CRYSOL, part of the ATSAS software package (version 3.1.0)86 (link),87 (link). The initial all-atom model of the full-length SH2/SH2 domain-swapped dimer was generated with PyMOL version 2.5.2 using PDB structures 1GRI (full-length GRB2 dimer) and 6ICH (SH2 domain-only dimer)60 (link),71 (link),108 . Each chain of the 1GRI dimer was superimposed over one of the SH2 domains comprising the 6ICH dimer. Intermodular linkers were built, N-terminal His-tags added, and missing amino acids inserted using YASARA109 (link). Reconstruction of the electron density was calculated from SAXS data using the program DENSS version 1.6 embedded in the BioXTAS RAW software89 (link). The final representative model was colorized and annotated using PyMOL and Chimera version 1.16110 (link).
Amino Acids
Buffers
Chimera
Electrons
GRB2 protein, human
Proteins
Radius
Reading Frames
Reconstructive Surgical Procedures
SH2 Domain
Guided by the cryo‐EM density map 1 (Appendix Fig S3 ) and XL‐MS distance restraints (Fig 2C ), a partial molecular model of E27 was generated de novo using the program Coot 0.9.5 (Emsley & Cowtan, 2004 (link); Emsley et al, 2010 (link)). In addition, in silico AlphaFold2 structure prediction of E27 was performed (Jumper et al, 2021 (link)) using the ColabFold web server (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb ; Mirdita et al, 2022 (link)). The AlphaFold2 output coordinates were in very good agreement with the experimentally determined E27 structure (RMSD of 1.489 Å, 2764 aligned atoms). Accordingly, the model was completed by merging the initial model with the AlphaFold2 template and adjusting backbone and side chain positions where necessary using Coot 0.9.5. DDB1 coordinates were taken from a previously solved DDB1/DCAF1‐CtD complex structure (Banchenko et al, 2021 (link)), rigid body‐fitted and adjusted manually using Coot 0.9.5. The coordinates of the rnSTAT2 CCD were extracted from the AlphaFold protein structure database (https://alphafold.ebi.ac.uk/entry/Q5XI26 ; Varadi et al, 2022 (link)), rigid body‐fitted and adjusted manually using Coot 0.9.5. As final step, automated real space refinement was performed using the program Phenix 1.19.2–4158 (Liebschner et al, 2019 (link)). The refined DDB1/E27/STAT2 CCD structure was then fitted as rigid body in cryo‐EM density map 2 (Appendix Fig S3 ). Full‐length rnSTAT2 coordinates (https://alphafold.ebi.ac.uk/entry/Q5XI26 ) were placed by structural superposition of the CCDs. The STAT2 NTD and TAD were removed, and flexible loops in the SH2 domain were trimmed, because they could not be assigned to the experimental density, indicating that they are flexibly attached and mobile relative to the other STAT2 domains. Lastly, a chain break was introduced between STAT2 residues 315 and 317, and the STAT2 portion encompassing residues 317–701 (DBD, LD and SH2) was once more separately fitted as rigid body, to account for a small movement relative to the CCD. Subsequently, the model was subjected to rigid body and grouped b‐factor real space refinement using Phenix 1.19.2–4158, followed by molecular dynamics flexible fitting using the Namdinator server (Kidmose et al, 2019 ), applying default parameters.
Complement Factor B
DDB1 protein, human
Human Body
MAP2 protein, human
Molecular Dynamics
Movement
Muscle Rigidity
SH2 Domain
STAT2 protein, human
Vertebral Column
VprBP protein, human
Plasmids for transient transfection of GFP only pEGFP-N1 (Clontech) or GFP-N-terminal fusions to AP180ct (rat aa 530‐915) and dynamin1_S45N have been described9 (link),11 (link). GFP-GPI was created by cloning the CD55 GPI-anchor sequence (aa 345-381) into pEGFP-N1. VAV1-3 C-terminally HA-tagged expression plasmids were from Addgene (#14553, #14554, #14554). All constructs described below were made by Gateway recombination (Thermo Fisher Scientific). HER2 constructs are based on cDNA reference sequence (NM_004448.4). Full-length HER2 (aa 1–1255) and a construct lacking the C-terminal cytoplasmic domain (aa 1–694) designated HER2_ΔCT, were C-terminally fused with the porcine teschovirus-1-derived P2A cleavage sequence (ATNFSLLKQAGDVEENPGP) followed by the blasticidin resistance cassette. All HER2-based constructs were recombined into a modified version of the pLenti-6-V5_Dest (Thermo Fisher Scientific V49610) in which the blasticidin resistance cassette had been exchange to puromycin. Trastuzumab non-binding mutants of HER2 (Tzmut) were engineered by deletion of aa 556-651 as previously described15 (link). VAV2 lacking the SH2 domain (aa 665–772) was generated by PCR mutagenesis of the VAV2 ORF amplified from addgene plasmid #14554. VAV2 dominant-negative constructs comprising residues 546–878 or lacking catalytic residues 341–347 were generated based on previously published data on VAV132 (link), by PCR mutagenesis of the VAV2 ORF amplified from Addgene plasmid #14554. Wt and mutant VAV2 constructs were recombined into a C-terminal monomeric-eGFP expression vector. Rac1 wt and dominant-negative mutant T17N as well as Ras dominant-negative mutant T17N were synthesised as codon-optimised geneblocks (Integrated DNA Technologies), based on cDNA reference sequences (NM_006908.5, Rac1 and NM_005343.4 hRas iso1) and recombined into an N-terminal monomeric-eGFP expression vector. The transferrin-receptor ectodomain (aa 89–760) was cloned into a baculovirus expression vector containing an N-terminal melittin signal peptide sequence (MKFLVNVALVFMVVYISYIYAA) and a C-terminal 3 C cleavage site followed by a 10x-His tag. All plasmids were verified by DNA sequencing.
Baculoviridae
Base Sequence
Catalysis
Cloning Vectors
Codon
Cytokinesis
Cytoplasm
Deletion Mutation
DNA, Complementary
ERBB2 protein, human
HRAS protein, human
Melitten
Mutagenesis
Peptides
Plasmids
Porcine teschovirus
Puromycin
Recombination, Genetic
SH2 Domain
Signal Peptides
TFRC protein, human
Transfection
Transients
Trastuzumab
VAV1 protein, human
Analysis was performed at the Protein Array and Analysis Core (MD Anderson Center, Houston, TX, USA). The peptide array used for the analysis contained the GST-tagged SH2 domains from 124 different proteins, each spotted in duplicate into eight grids. GST alone is present in the middle of each grid as a negative control. To generate the array, the SH2 domains were cloned into the pGEX-4T1 vector and synthesized by the Biomatik Corporation (Kitchener, Canada). Expression and purification of recombinant proteins were carried out essentially as previously described [20 (link)]. The GST-tagged constructs, and GST alone, were then spotted onto nitrocellulose-coated glass slides using the Aushon 2470 Microarrayer (Quanterix) (Grace Bio-Labs, Bend, OR, USA). Equal loading of the constructs was verified by probing the array with an anti-GST primary antibody and fluorescent dye-conjugated secondary antibodies.
Biotinylated IQGAP1 peptides (amino acids 1502 to 1518) with either non-phosphorylated or phosphorylated Tyr1510 were synthesized by Thermo Fisher Scientific. The peptides were conjugated to streptavidin-Cy3 fluorophore for use as microarray probes. After incubation for 1 h at 22 °C in blocking buffer (PBS-Tween containing 3% milk and 3% BSA), the arrays were incubated with fluorophore-conjugated peptides for 16 h at 4 °C in blocking buffer. The microarrays were washed with PBS-Tween, dried, and fluorescence from the bound peptides was detected using the InnoScan 1100 AL Fluorescence Scanner (Innopsys, Chicago, IL, USA) [20 (link)].
Biotinylated IQGAP1 peptides (amino acids 1502 to 1518) with either non-phosphorylated or phosphorylated Tyr1510 were synthesized by Thermo Fisher Scientific. The peptides were conjugated to streptavidin-Cy3 fluorophore for use as microarray probes. After incubation for 1 h at 22 °C in blocking buffer (PBS-Tween containing 3% milk and 3% BSA), the arrays were incubated with fluorophore-conjugated peptides for 16 h at 4 °C in blocking buffer. The microarrays were washed with PBS-Tween, dried, and fluorescence from the bound peptides was detected using the InnoScan 1100 AL Fluorescence Scanner (Innopsys, Chicago, IL, USA) [20 (link)].
Amino Acids
Antibodies
Antibodies, Anti-Idiotypic
Buffers
Cloning Vectors
Decompression Sickness
Fluorescence
Fluorescent Dyes
Microarray Analysis
Milk, Cow's
Nitrocellulose
Peptides
Protein Arrays
Protein Domain
Recombinant Proteins
SH2 Domain
Streptavidin
Tweens
The plasmids coding for the GST-tagged SH2 domains of Abl1 and Abl2 were kindly provided by the Protein Array and Analysis Core (MD Anderson Center, Houston, TX, USA). These constructs and GST-IQGAP1 were expressed in Escherichia coli essentially as previously described [18 (link)]. Briefly, induction of expression was carried out with 100 μM iso-propyl-β-D-thiogalactoside at 37 °C. After 3 h, bacteria were lysed by sonication in PBS (pH 7.2) containing 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mM dithiothreitol (DTT). After removal of cell debris (15,000× g for 10 min at 4 °C), lysates were applied onto glutathione-Sepharose columns. The columns were washed with PBS containing 10 mM DTT before recovery of the beads. The purity and molecular masses of the GST fusion proteins were examined by heating the samples bound to the beads for 5 min at 95 °C in Laemmli sample buffer for subsequent SDS-PAGE and Coomassie blue staining.
Bacteria
Cells
Coomassie blue
Dithiothreitol
Edetic Acid
Escherichia coli
Glutathione
Laemmli buffer
Phenylmethylsulfonyl Fluoride
Plasmids
Protein Arrays
Proteins
SDS-PAGE
Sepharose
SH2 Domain
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RFP-Trap is a laboratory product designed to capture and purify mCherry- or RFP-tagged proteins from cell lysates. It consists of agarose beads coated with a high-affinity RFP-binding protein that can efficiently pull down RFP- or mCherry-fusion proteins for further analysis.
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