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

• Published in: Nature communications 2024-10, Vol.15 (1), p.8885-14, Article 8885
• Main Authors: Kroupova, Alena, Spiteri, Valentina A., Rutter, Zoe J., Furihata, Hirotake, Darren, Darren, Ramachandran, Sarath, Chakraborti, Sohini, Haubrich, Kevin, Pethe, Julie, Gonzales, Denzel, Wijaya, Andre J., Rodriguez-Rios, Maria, Sturbaut, Manon, Lynch, Dylan M., Farnaby, William, Nakasone, Mark A., Zollman, David, Ciulli, Alessio
• Format: Article
• Language: English
• Subjects: 631/154/309/2420; 631/45/474/2073; 631/535/1266; 631/92/469; 82/103; 82/80; 82/83; Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Biodegradation; Crystallization; Crystallography; Crystallography, X-Ray; Design; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Drug development; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Glues; High resolution; Humanities and Social Sciences; Humans; Lenalidomide; Models, Molecular; multidisciplinary; Protein Conformation; Protein structure; Proteins; Proteolysis; Science; Science (multidisciplinary); Thalidomide; Thalidomide - analogs & derivatives; Thalidomide - chemistry; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-52871-9

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. Structure-based drug discovery of cereblon (CRBN)-recruiting protein degraders has been to date challenging due to limitations with current constructs for recombinant protein expression. In this work, the authors design and validate a truncated CRBN construct, CRBNmidi, that enables crystallization and biophysical characterization of CRBN-binding ligands and degraders.