# Characterizing the Monomer–Dimer Equilibrium of UbcH8/Ube2L6: A Combined SAXS and NMR Study

## TL;DR

This study uses SAXS and NMR to determine that UbcH8 can exist as a monomer or dimer, with dimerization affecting its functional interfaces.

## Contribution

The study reveals a concentration-dependent monomer–dimer equilibrium of UbcH8 and identifies a dimerization interface using SAXS and NMR.

## Key findings

- SAXS shows UbcH8 forms a dimer that can dissociate when fused to GST.
- NMR confirms a concentration-dependent monomer–dimer equilibrium.
- Dimerization induces conformational changes at E1 and E3 interfaces.

## Abstract

Interferon-stimulated gene-15 (ISG15) is an interferon-induced
protein with two ubiquitin-like (Ubl) domains linked by a short peptide
chain and is a conjugated protein of the ISGylation system. Similar
to ubiquitin and other Ubls, ISG15 is ligated to its target proteins
through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8,
and HERC5, respectively. Ube2L6/UbcH8 plays a central role in ISGylation,
underscoring it as an important drug target for boosting innate antiviral
immunity. Depending on the type of conjugated protein and the ultimate
target protein, E2 enzymes have been shown to function as monomers,
dimers, or both. UbcH8 has been crystallized in both monomeric and
dimeric forms, but its functional state remains unclear. Here, we
used a combined approach of small-angle X-ray scattering (SAXS) and
nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s
oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure
that could be dissociated when fused N-terminally to glutathione S-transferase.
NMR spectroscopy validated the presence of a concentration-dependent
monomer–dimer equilibrium and suggested a back-side dimerization
interface. Chemical shift perturbation and peak intensity analysis
further suggest dimer-induced conformational dynamics at the E1 and
E3 interfaces, providing hypotheses for the protein’s functional
mechanisms. Our study highlights the power of combining NMR and SAXS
techniques to provide structural information about proteins in solution.