Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• MYCBP2 is a crucial ubiquitin E3 ligase for neurodevelopment and axon maintenance.
• It utilizes a unique RING-Cys-Relay (RCR) mechanism for ubiquitin transfer to non-lysine substrates.
• The molecular mechanisms of E2-E3 ubiquitin transfer and relay, and their link to neural functions, remain largely unknown.

Purpose of the Study:

• To elucidate the structural and mechanistic basis of ubiquitin transfer and relay by MYCBP2.
• To investigate the functional significance of the RCR mechanism and MYCBP2's enzymatic activity in neural development and axon maintenance.

Main Methods:

• Crystal structure determination of a covalently trapped E2~Ub:MYCBP2 transfer intermediate.
• Biochemical assays to analyze ubiquitin transfer and relay mechanisms.
• Generation and analysis of RCR-defective knock-in mouse models.

Main Results:

• The crystal structure revealed key rearrangements during E2-E3 ubiquitin transfer and Ub relay.
• Ubiquitin transfer to the upstream cysteine requires a specific E2~Ub conjugate conformation.
• Ubiquitin relay involves a helix-coil transition, and RCR defects lead to neurodevelopmental defects and impaired axon degeneration.

Conclusions:

• The study provides structural insights into the MYCBP2 RCR mechanism, linking E2-E3 ubiquitin transfer and relay.
• MYCBP2's substrate esterification activity and the RCR mechanism are essential for normal neural development and programmed axon degeneration.