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Updated: May 18, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Structure of the HECT C-lobe of the UBR5 E3 ubiquitin ligase

Edna Matta-Camacho1, Guennadi Kozlov, Marie Menade

  • 1Groupe de Recherche axé sur la Structure des Protéines, Department of Biochemistry, McGill University, Montreal, Quebec H3G 0B1, Canada.

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
|October 3, 2012
PubMed

Insights

The crystal structure of human UBR5

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • UBR5 (also known as EDD, Rat100, or hHYD) is a HECT-class E3 ubiquitin ligase.
  • HECT domain E3 ligases mediate ubiquitination through a cascade involving E2 enzymes.
  • The UBR5 HECT domain has distinct N- and C-lobes.

Purpose of the Study:

  • To determine the high-resolution crystal structure of the C-lobe of the human UBR5 HECT domain.
  • To investigate structural features unique to UBR5 compared to other HECT domains.
  • To explore the functional implications of these structural features for UBR5 ubiquitination activity.

Main Methods:

  • X-ray crystallography to determine the high-resolution structure of the UBR5 HECT domain C-lobe.
  • Ubiquitination assays to assess E3-ubiquitin complex formation.
  • Nuclear Magnetic Resonance (NMR) experiments to investigate protein-protein interactions.

Main Results:

  • The crystal structure revealed unique features, including a four-residue insert in the second helix of the C-lobe.
  • This insert alters the orientation of a preceding loop, potentially influencing E2 enzyme specificity.
  • Ubiquitination assays confirmed the C-lobe's ability to form a thioester-linked E3-ubiquitin complex.
  • NMR experiments did not show physical interaction between the UBR5 C-lobe and UBCH4.

Conclusions:

  • The unique structural elements of the UBR5 HECT domain C-lobe may contribute to its specificity towards the E2 enzyme UBCH4.
  • Despite functional assays, direct physical interaction between the UBR5 C-lobe and UBCH4 was not observed.
  • This study enhances understanding of UBR5's structural basis for ubiquitination activity.

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