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

TY - JOUR

T1 - Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

AU - Kroupova, Alena

AU - Spiteri, Valentina

AU - Rutter, Zoe

AU - Furihata, Hirotake

AU - Darren, Darren

AU - Ramachandran, Sarath

AU - Chakraborti, Sohini

AU - Haubrich, Kevin

AU - Pethe, Julie

AU - Gonzales, Denzel

AU - Wijaya, Andre

AU - Rodriguez-Rios, Maria

AU - Sturbaut, Manon

AU - Lynch, Dylan

AU - Farnaby, William

AU - Nakasone, Mark

AU - Zollman, David

AU - Ciulli, Alessio

N1 - Publisher Copyright: © The Author(s) 2024.

PY - 2024/10/15

Y1 - 2024/10/15

N2 - The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBN midi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBN midi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBN midi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

AB - The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBN midi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBN midi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBN midi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.

KW - Ubiquitin-Protein Ligases/metabolism

KW - Humans

KW - Crystallography, X-Ray

KW - Adaptor Proteins, Signal Transducing/metabolism

KW - Proteolysis

KW - Escherichia coli/metabolism

KW - Models, Molecular

KW - Thalidomide/chemistry

KW - Lenalidomide

KW - Protein Conformation

KW - DNA-Binding Proteins/metabolism

UR - https://www.scopus.com/pages/publications/85206506600

U2 - 10.1038/S41467-024-52871-9

DO - 10.1038/S41467-024-52871-9

M3 - Article

C2 - 39406745

SN - 2041-1723

VL - 15

JO - Nature Communications

JF - Nature Communications

M1 - 8885

ER -