Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

Duarte D, Lamontanara A, La Sala G, Jeong S, Sohn Y, Panjkovich A, Georgeon S, Kükenshöner T, Marcaida M, Pojer F, De Vivo M, Svergun D, Kim H, Dal Peraro M, Hantschel O, (2019) DOI

SASDF63 – Bruton's Tyrosine Kinase - SH2-kinase domain

Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain
MWexperimental 47 kDa
MWexpected 46 kDa
VPorod 64 nm3
log I(s) 8.51×101 8.51×100 8.51×10-1 8.51×10-2
Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain small angle scattering data  s, nm-1
ln I(s)
Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain Guinier plot ln 8.51×101 Rg: 2.8 nm 0 (2.8 nm)-2 s2
(sRg)2I(s)/I(0)
Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain Kratky plot 1.104 0 3 sRg
p(r)
Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain pair distance distribution function Rg: 2.9 nm 0 Dmax: 9.4 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Bruton's Tyrosine Kinase - SH2-kinase domain Rg histogram Rg, nm

log I(s)
 s, nm-1
Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain DAMMIN model

Synchrotron SAXS data for SH2-kinase domain (amino acids 270-659) wild-type of human Bruton’s tyrosine kinase (Btk) in 20 mM Tris-HCl, 150 mM NaCl, 5% Glycerol, 1 mM TCEP, pH 7.5 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Dectris Pilatus 1M detector at a sample-detector distance of 2.849 m and at a wavelength of λ = 0.099 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size exclusion chromatography (SEC) SAXS was performed by injecting 100µl of protein sample at 20mg/mL in a Superdex S200 Increase 10/300 column (GE Healthcare) at flow rate of 0.7mL/minute at room temperature. 2100 frames were collect during the 35 minutes total run. SEC-SAXS data frames 1031 - 1077 corresponding to monomeric peak were scaled, averaged and subtracted from an appropriate the solvent-blank to produce the final curve. The final scattering curve was analysed using PRIMUS software (ATSAS). The bead model depicts the DAMMIN ab initio reconstruction. Ensemble optimisation method (EOM) was performed using SH2 and kinase domain structures (2GE9 and 1K2P, PDB) to assess construct’s flexibility.

Bruton's tyrosine kinase - Src homolgy domain 2 - kinase domain (hBtk - SH2-KD)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   45.5 kDa
Sequence   FASTA