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Covalently Linked Protein Regulators

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.

Protein Complexes with Interchangeable Parts

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...

The Proteasome

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...

Regulated Protein Degradation

Regulated Protein Degradation

It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...

The Proteasome

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin...

The Proteasome Structure

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...

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Related Experiment Video

Updated: Jul 13, 2026

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.

Danny T Huang¹, Harold W Hunt, Min Zhuang

¹Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

|January 16, 2007

Summary

The study reveals how ubiquitin-like protein (UBL) conjugation enzymes switch binding affinities through a thioester mechanism. This conformational change drives sequential steps in UBL modification pathways.

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Area of Science:

Biochemistry
Structural Biology
Molecular Cell Biology

Background:

Ubiquitin-like proteins (UBLs) are modified through dynamic E1-E2-E3 enzyme cascades.
E1 enzymes activate UBLs via adenylation, forming thioester intermediates crucial for subsequent conjugation steps.

Purpose of the Study:

To elucidate the structural mechanism of UBL activation and transfer within the human NEDD8 pathway.
To understand how enzyme-substrate interactions and conformational changes drive UBL conjugation cascades.

Main Methods:

Reported structural analysis of a trapped UBL activation complex.
Utilized a complex containing the NEDD8 E1 enzyme (APPBP1-UBA3), two NEDD8 molecules, an inactive E2 enzyme (Ubc12), and MgATP.

Main Results:

Identified a 'thioester switch' mechanism that modulates E1-E2 affinities.
Revealed two E2 binding sites dependent on NEDD8's thioester linkage to E1, one unmasked by E1 conformational change.
Demonstrated that NEDD8 transfer to E2 induces E1 conformational reversion, facilitating product release.

Conclusions:

The transfer of thioester linkages between conjugation enzymes drives conformational changes and alters interaction networks.
This mechanism is essential for propelling consecutive steps in UBL conjugation cascades.