Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Sequence determinants of E2-E6AP binding affinity and specificity.

Ziad M Eletr1, Brian Kuhlman

  • 1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.

Journal of Molecular Biology
|April 17, 2007
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Enhancing machine learning-based binder design with high-throughput screening: A comparison of mRNA and yeast display technologies.

Protein science : a publication of the Protein Society·2026
Same author

Deep learning based design of buried hydrogen bond networks with HBDesigner.

bioRxiv : the preprint server for biology·2026
Same author

De novo masking domains that gate TNF-family ligand assembly and activity.

bioRxiv : the preprint server for biology·2026
Same author

Intensity modulation of trichromatic split fluorescent proteins for live cell mapping.

Cell reports methods·2026
Same author

Enhancing Enzymatic Bioconjugation Efficiency via Computer-Based Installation of a Substrate Recruitment Domain.

Bioconjugate chemistry·2026
Same author

Enhancing ML-based binder design with high-throughput screening: a comparison of mRNA and yeast display technologies.

bioRxiv : the preprint server for biology·2026

Ubiquitin conjugation involves E1, E2, and E3 enzymes. Researchers studied E2-E3 interactions using a fluorescence polarization assay, finding that both promoting and preventing interactions dictate binding specificity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Ubiquitin conjugation is a crucial post-translational modification mediated by E1, E2, and E3 enzymes.
  • Specific interactions between E2 conjugating enzymes and E3 ligases determine substrate ubiquitination.
  • E6AP, a HECT-family E3 ligase, interacts with E2 enzymes UbcH7 and UbcH8.

Purpose of the Study:

  • To identify sequence determinants governing E2-E3 binding specificity.
  • To quantify the binding affinity between E2 and E3 enzymes using a novel assay.

Main Methods:

  • Development of a quantitative fluorescence polarization assay to measure E2-E3 binding affinity.
  • Alanine scanning mutagenesis of the UbcH7-E6AP interface to identify key binding residues.
  • Site-directed mutagenesis of UbcH5b to investigate the role of specific residues in E6AP binding.

Related Experiment Videos

Main Results:

  • Identified critical residues on both UbcH7 and E6AP that contribute significantly to binding free energy.
  • Mutations at conserved 'hot spot' residues in UbcH5b did not enhance E6AP binding as expected.
  • A mutation at a non-hot spot residue in UbcH5b unexpectedly increased its affinity for E6AP.

Conclusions:

  • E2-E3 binding specificity is determined by a complex interplay of both favorable and unfavorable interactions.
  • Unforeseen interactions at non-interface residues can significantly influence E2-E3 binding affinity and specificity.