GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production.

Sandouk A, Xu Z, Baruah S, Tremblay M, Hopkins JB, Chakravarthy S, Gakhar L, Schnicker NJ, Houtman JCD, Sci Rep 13(1):3505 (2023) Europe PMC

SASDP58 – Growth factor receptor-bound protein 2 (GRB2) - N188D/N214D mutant dimer

Growth factor receptor-bound protein 2 (N188D, N214D)
MWexperimental 65 kDa
MWexpected 55 kDa
VPorod 140 nm3
log I(s) 2.66×100 2.66×10-1 2.66×10-2 2.66×10-3
Growth factor receptor-bound protein 2 (N188D, N214D) small angle scattering data  s, nm-1
ln I(s)
Growth factor receptor-bound protein 2 (N188D, N214D) Guinier plot ln 2.67×100 Rg: 4.1 nm 0 (4.1 nm)-2 s2
(sRg)2I(s)/I(0)
Growth factor receptor-bound protein 2 (N188D, N214D) Kratky plot 1.104 0 √3 sRg
p(r)
Growth factor receptor-bound protein 2 (N188D, N214D) pair distance distribution function Rg: 4.2 nm 0 Dmax: 15 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Growth factor receptor-bound protein 2 (N188D, N214D) PYMOL model
log I(s)
 s, nm-1
Growth factor receptor-bound protein 2 (N188D, N214D) PYMOL model
SEC-MALS-SAXS data sets were collected using the 18-ID-D BioCAT Beamline at the Advanced Proton Source (APS) at Argonne National Laboratory (Chicago, IL). Samples were centrifuged for 5 min at 13,000 rpm to remove any potential aggregates prior to column loading. Samples containing 4-9 mg/mL of GRB2 WT or mutants in 250 μL were injected onto a 24-mL Superdex 75 Increase 10/300 analytical-grade column (GE) equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl, and 1 mM DTT at a flow rate of 0.5 mL/minute on an Agilent 1300 chromatography system. Column eluant was analyzed in line by the UV absorbance detector of the Agilent 1300 chromatography system, then subsequently directed into the DAWN Heleos-II light scattering (LS) and OptiLab T-rEX refractive index detectors in series. Finally, the elution trajectory directed samples into a 1.0-mm ID quartz capillary SAXS sample cell. Scattering data were collected every 1 sec using a 0.5-sec exposure and detected with a Pilatus 3 1M pixel detector (DECTRIS) with a 12 KeV (0.1033 nm wavelength) X-ray beam covering an s-range of 0.045 < s < 3.5 nm-1 (s = 4π/λsinθ, where λ is the wavelength and 2θ is the scattering angle). Accurate protein molecular weights from MALS data were determined using the ASTRA software (Wyatt Technology).

Growth factor receptor-bound protein 2 (N188D, N214D) (GRB2- N188D/N214D)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dimer
Mon. MW   27.7 kDa
 
UniProt   P62993 (1-217)
Sequence   FASTA
 
PDB ID   1GRI