Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

Kroupova A, Spiteri V, Rutter Z, Furihata H, Darren D, Ramachandran S, Chakraborti S, Haubrich K, Pethe J, Gonzales D, Wijaya A, Rodriguez-Rios M, Sturbaut M, Lynch D, Farnaby W, Nakasone M, Zollman D, Ciulli A, Nature Communications 15(1) (2024) DOI

SASDU62 – Cereblon-midi (CRBNmidi), an engineered Cereblon construct for crystallographic and biophysical studies, bound to Mezigdomide

Cereblon-midi
mezigdomide
MWexperimental 37 kDa
MWexpected 38 kDa
VPorod 60 nm3
log I(s) 1.62×10-2 1.62×10-3 1.62×10-4 1.62×10-5
Cereblon-midi mezigdomide small angle scattering data  s, nm-1
ln I(s)
Cereblon-midi mezigdomide Guinier plot ln 1.62×10-2 Rg: 2.3 nm 0 (2.3 nm)-2 s2
(sRg)2I(s)/I(0)
Cereblon-midi mezigdomide Kratky plot 1.104 0 3 sRg
p(r)
Cereblon-midi mezigdomide pair distance distribution function Rg: 2.3 nm 0 Dmax: 7.3 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Cereblon-midi mezigdomide PYMOL model

log I(s)
 s, nm-1
Cereblon-midi mezigdomide SWISSMODEL model

Synchrotron SAXS data from solutions of Cereblon-midi (CRBNmidi), an engineered Cereblon construct for crystallographic and biophysical studies, bound to Mezigdomide in 20 mM Hepes, 500 mM NaCl, 0.5 mM TCEP, pH 7.5 were collected on the B21 beam line at the Diamond Light Source storage ring (Didcot, UK) using a Eiger 4M detector at a sample-detector distance of 3.7 m and at a wavelength of λ = 0.09464 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 50.00 μl sample at 6.5 mg/ml was injected at a 0.08 ml/min flow rate onto a Cytiva Superdex 200 Increase 3.2/300 column at 15°C. 600 successive 3 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC-SAXS of CRBN-midi in complex with mezigdomide (PubChem: https://pubchem.ncbi.nlm.nih.gov/compound/137379043, ChEMBL: https://www.ebi.ac.uk/chembl/compound_report_card/CHEMBL4648616/). Before SEC, CRBN-midi (6.5 mg/ml, 175 µM) were incubated with 400 µM mezigdomide in SEC buffer with 2 % DMSO.

Cereblon-midi (CRBNmidi)
Mol. type   Protein
Olig. state   Monomer
Mon. MW   37.4 kDa
Sequence   FASTA
 
mezigdomide (CC-92480)
Mol. type   Other
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   0.6 kDa
Chemical formula