Related Experiment Video

Updated: Jul 1, 2026

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
10:27

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Published on: December 5, 2019

Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2)

Peter D Mace1, Katrin Linke, Rebecca Feltham

  • 1Biochemistry Department, University of Otago, Dunedin 9054, New Zealand.

The Journal of Biological Chemistry
|September 12, 2008
PubMed

Insights

Inhibitor of apoptosis (IAP) proteins regulate cell death and are targeted in cancer. Their ubiquitylation and degradation are key to cell death induction, involving the RING domain. This study reveals structural insights into IAP regulation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Inhibitor of apoptosis (IAP) proteins are crucial negative regulators of programmed cell death.
  • High expression of IAPs is observed in numerous cancers, making them therapeutic targets.
  • Antagonists targeting IAPs induce cell death via ubiquitylation and degradation of these proteins.

Purpose of the Study:

  • To elucidate the structural mechanisms underlying the regulation of cellular inhibitor of apoptosis (cIAP) proteins.
  • To investigate the role of the RING domain, E2 enzyme binding, and dimer formation in IAP function and stability.
  • To understand how these structural features influence IAP protein abundance and sensitivity to antagonists.

Main Methods:

  • X-ray crystallography was used to determine the structures of the cIAP2 RING domain homodimer alone and bound to the UbcH5b E2 enzyme.
  • Site-directed mutagenesis was employed to probe the function of residues at the E2-binding surface and the C-terminal region.
  • In vivo experiments were conducted to assess the stability of IAP proteins upon disruption of E2 binding or dimerization.

Main Results:

  • Crystal structures revealed conformational changes in the cIAP2 RING domain upon binding to the UbcH5b E2 enzyme.
  • Autoubiquitylation, dependent on E2-binding surface residues, is essential for regulating IAP protein abundance.
  • Dimer formation of the RING domain is critical for E3 ligase activity; mutations disrupting dimerization diminished this activity.
  • Disrupting either E2 binding or dimerization stabilized IAP proteins against antagonists in vivo.

Conclusions:

  • The cIAP2 RING domain undergoes structural adaptations upon E2 enzyme binding, influencing its ligase activity.
  • Both E2 enzyme interaction and homodimerization of the RING domain are indispensable for proper IAP function, including autoubiquitylation and regulation of protein levels.
  • Targeting E2 binding or dimerization represents a potential strategy to stabilize IAP proteins and enhance cancer cell death induced by IAP antagonists.

More Related Videos

In Vitro Analysis of E3 Ubiquitin Ligase Function06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale10:50

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Published on: March 14, 2019

Related Concept Videos

Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
The Proteasome Structure01:17

The Proteasome Structure

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an...
Coat Assembly and GTPases01:33

Coat Assembly and GTPases

Vesicles incorporate different coat protein subunits in different cell locations, which changes the properties of the coat, such as the shape and geometry of the transport vesicles. Thus, vesicle coat proteins also play a significant role in cargo selection.
Coat assembly depends on the local availability of phosphatidylinositol phosphates or PIPs and GTP-binding proteins. Adaptor proteins, which link the coat proteins to the membrane, bind to these PIPs and play a crucial role in controlling...
Anaphase Promoting Complex00:50

Anaphase Promoting Complex

The stepwise destruction of specific proteins is necessary for the progression and completion of the cell cycle. Such proteins are ubiquitinated by ubiquitin ligases and then subsequently destroyed by the proteasome. The SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC) are two important ubiquitin ligases involved in cell cycle progression. While SCF is active throughout the cell cycle, APC gets activated during metaphase to anaphase transition. Cdc20 or Cdh1 binds to APC and...
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...