UC San Diego

Since the development of cereblon (CRBN) as proteolysis targeting chimeric (PROTAC), targeted protein degradation (TPD) has become the new paradigm in therapeutic design. Currently TPD has a limited number of E3 engagers, the small diversity leading to potential common off-target effects. Dr. Yuan Chen’s research group has developed ligands for the UBR E3 ligases with an affinity of Kd<1μM. This project focused on elucidating the structure of these ligands bound to the UBR-box domain of human UBR2 protein via x-ray crystallography. Three structures were resolved. UBR2 crystal was grown in 0.1M Bis-Tris pH 5.5 28% PEG 4000 for 7 days. UBR2-AF27 crystal was grown in 0.1M HEPES pH 7.0 26% PEG 4000 for 3 days. UBR2-AF23 crystal was grown in 0.1M HEPES pH 7.0 27% PEG 4000 for 3 days. Diffraction data sets were collected remotely at Stanford

Synchrotron Radiation Light source on a Dectris CCD PILATUS 6M detector on beamline BL12-2(λ = 0.97946Å), with beam size of 50.0 x 15.0 μm and energy at 12658.000eV with transmission of 10.602%. The resulting ligand bound structures deviate from docking simulations. While the Arg recognizing binding pocket behaved as expected, the resulting structures show insight on how bulky hydrophobic groups were bound to the UBR-box of UBR2. Gly144, Gly145, Gly146, and Gly147 (the 4G pocket) display flexibility, stabilize the ligand via hydrogen bonding and house the bulky hydrophobic residue as opposed to the previously reported hydrophobic pocket.