RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• The anaphase-promoting complex/cyclosome (APC) is a crucial E3 ubiquitin ligase controlling cell division.
• Disordered substrates are recruited to the APC via specific motifs and ubiquitinated by the APC/APC11-UBCH10 catalytic core.
• Mechanisms of E2 enzyme (UBCH10) recruitment and substrate ubiquitination by the APC remain poorly understood.

Purpose of the Study:

• To elucidate the structural mechanisms of UBCH10 recruitment to the APC(CDH1) complex.
• To understand how the APC facilitates ubiquitination of intrinsically disordered substrates.
• To provide the first structural view of a cullin-RING E3 ligase with E2 and substrate juxtaposed.

Main Methods:

• Cryo-electron microscopy (cryo-EM) of a trapped APC(CDH1)-UBCH10∼Ub-substrate intermediate.
• X-ray crystallography of isolated APC2 and UBCH10 interactions.
• Biochemical assays to study ubiquitination mechanisms.

Main Results:

• UBCH10 (UBE2C) is recruited to the APC not only by APC11 but also via interactions with APC2, forming a tripartite complex.
• Structural data reveals the juxtaposition of the E3 ligase, E2 enzyme, and substrate.
• Tripartite interactions dictate UBCH10 specificity and enable efficient ubiquitination of accessible lysines on disordered substrates.

Conclusions:

• The APC utilizes tripartite interactions involving APC2, APC11, and UBCH10 to achieve substrate specificity and catalytic efficiency.
• These interactions stabilize the dynamic catalytic module, enhance collision probability with substrates, and facilitate regulation of cell division.
• This study provides unprecedented structural insights into the ubiquitination process mediated by cullin-RING E3 ligases.