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Related Concept Videos

Conserved Binding Sites01:49

Conserved 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...
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.
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Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

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Regulated Protein Degradation

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The Proteasome01:13

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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...

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Updated: Jun 14, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
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Published on: May 14, 2021

Dynamic Hotspots in the Uba7 Ubiquitin-Fold Domain Direct UbcH8 Recognition.

Çağdaş Dağ1,2,3,4, Mahil Lambert1,4, Alp E Kazar1

  • 1Nanofabrication and Nanocharacterization Center for Scientific and Technological Advanced Research (n2STAR), Koç University, İstanbul 34450, Turkiye.

Biochemistry
|March 3, 2026
PubMed
Summary

The study reveals how the Uba7 ubiquitin-fold domain (UFD) structurally interacts with the UbcH8 enzyme, clarifying a key step in interferon-stimulated gene 15 (ISG15) conjugation for innate immunity.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Immunology

Background:

  • ISGylation is a critical ubiquitin-like modification in innate immunity.
  • The E1-E2 enzyme interaction in ISGylation, specifically Uba7-UbcH8, is structurally undefined.
  • Understanding this interaction is key to deciphering innate immune signaling pathways.

Purpose of the Study:

  • To determine the solution NMR structure of the human Uba7 ubiquitin-fold domain (UFD).
  • To characterize the structural and dynamic basis of Uba7-UFD recognition by the E2 enzyme UbcH8.
  • To identify key residues and structural features governing E1-E2 binding specificity.

Main Methods:

  • Solution Nuclear Magnetic Resonance (NMR) spectroscopy, including chemical shift perturbation and 15N relaxation measurements.
  • Site-directed mutagenesis to probe critical residues and structural elements.
  • Functional characterization of Uba7-UFD and UbcH8 interactions.

Main Results:

  • The Uba7-UFD adopts a flexible ubiquitin-fold structure.
  • NMR and mutagenesis identified the UbcH8 interaction surface on Uba7-UFD.
  • Conformational dynamics of the UFD, particularly an acidic loop, are crucial for efficient UbcH8 binding.
  • Cysteine 996 is essential for UFD structural integrity and binding.

Conclusions:

  • The study provides a structural and dynamic model for E1-E2 recognition in ISGylation.
  • Conformational flexibility and specific loop architecture of the Uba7-UFD are vital for UbcH8 recruitment.
  • These findings offer insights into the regulation of innate immune signaling via ISGylation.