E2 variants for probing E3 ubiquitin ligase activities

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• E3 ligases and E2 enzymes are crucial for eukaryotic biology.
• The large number of E3 ligases (>600) and E2 enzymes (~40) in humans necessitates E2 enzymes co-functioning with multiple E3 ligases.
• Understanding these complex interactions is vital for deciphering cellular processes.

Purpose of the Study:

• To develop a method for interrogating specific E3 ligase pathways.
• To create E2 variants (E2Vs) with enhanced affinity and specificity for RING-between-RING (RBR) E3 ligases.
• To enable detailed mechanistic and structural analysis of E3 ligase functions.

Main Methods:

• Screening of phage-displayed libraries of structure-based E2 variants (E2Vs).
• Focus on RBR E3 ligases and UBE2L3/UBE2D-family E2 enzymes.
• Utilizing cryogenic electron microscopy (cryo-EM) for structural analysis.

Main Results:

• Discovery of E2Vs with enhanced affinity and specificity for multiple RBR E3 ligases (ARIH1, ARIH2, ANKIB1, CUL9, HOIL1, HOIP, RNF14).
• Successful differentiation of functions between co-functioning E3 ligases.
• Obtained high-resolution cryo-EM structures of RBR E3 ligase complexes.
• Profiled endogenous RBR E3 ligase responses to extracellular stimuli.

Conclusions:

• E2V technology provides a generalizable tool for in-depth analysis of E3 ligase functions.
• This approach facilitates mechanistic and structural studies of E3 ligase pathways.
• E2Vs aid in mapping E3 ligase activity states and protein interactions in cellular signaling.