Related Experiment Video
Updated: Jun 17, 2026
10:27
Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
Structural and functional characterization of the monomeric U-box domain from E4B.
Kyle A Nordquist1, Yoana N Dimitrova, Peter S Brzovic
1Department of Biochemistry, Vanderbilt University, Nashville,Tennessee 37232, USA.
Biochemistry
|December 19, 2009
Summary
The E4B U-box domain (E4BU) functions as a monomer, unlike most E3 ubiquitin ligases. Biophysical and NMR analyses reveal its unique structure and E2 enzyme interaction, maintaining ligase activity.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Genetics
Background:
- Oligomerization is crucial for E3 ubiquitin ligase function.
- Most E3 ligases function as dimers or higher-order oligomers.
- Yeast Ufd2 and mammalian E4B are exceptions, potentially acting as monomers.
Purpose of the Study:
- To determine the high-resolution NMR solution structure of the E4B U-box domain (E4BU).
- To investigate the biophysical properties and dimerization potential of E4BU.
- To map the interaction site between E4BU and the E2 conjugating enzyme UbcH5c.
Main Methods:
- Subcloning, overexpression in E. coli, and purification of E4BU.
- High-resolution Nuclear Magnetic Resonance (NMR) spectroscopy for structural determination.
- Biophysical analyses including interaction mapping with UbcH5c.
Main Results:
- E4BU is a stable monomeric protein adopting a U-box fold similar to homodimers.
- Sequence substitutions were identified that inhibit E4BU dimerization.
- NMR mapping revealed an E2 binding site on E4BU similar to dimeric ligases.
- E4BU demonstrated activity in autoubiquitination assays despite its monomeric state.
Conclusions:
- E4BU functions as an active monomer, challenging the paradigm of E3 ligase oligomerization.
- Structural insights into E4BU provide a basis for understanding monomeric E3 ligase function.
- The findings contribute to the broader understanding of U-box and RING domain E3 ligase mechanisms.
Related Concept Videos
01:57Protein 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...
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...
01:14Rab Proteins
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
02:21Globular and Fibrous Proteins
Many proteins can be classified into two distinct subtypes - globular or fibrous. These two types differ in their shapes and solubilities.
Globular proteins are also known as spheroproteins and typically are approximately round in shape. They contain a mix of amino acid types and contain differing sequences in their primary structures. Globular proteins have many different functions, such as enzymes, cellular messengers, and molecular transporters. These roles often require the proteins to be...
Globular proteins are also known as spheroproteins and typically are approximately round in shape. They contain a mix of amino acid types and contain differing sequences in their primary structures. Globular proteins have many different functions, such as enzymes, cellular messengers, and molecular transporters. These roles often require the proteins to be...
02:26Conservation of Protein Domains Over Different Proteins
Protein domains are small structurally independent units that are part of a single amino acid chain. Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
01:57Gene Families
Gene families consist of groups of genes proposed to have originated from a common ancestor. Typically these arise through events in which a gene or genes are mistakenly duplicated during cell division. Unlike their parent genes (which are subject to selection pressure to maintain function), these gene copies do not need to preserve their sequences and may evolve at a relatively faster rate.
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...
01:49Conserved Binding Sites
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...