Characterizing\nthe Monomer–Dimer Equilibrium\nof UbcH8/Ube2L6: A Combined SAXS and NMR Study

Interferon-stimulated gene-15 (ISG15) is an interferon-induced\nprotein with two ubiquitin-like (Ubl) domains linked by a short peptide\nchain and is a conjugated protein of the ISGylation system. Similar\nto ubiquitin and other Ubls, ISG15 is ligated to its target proteins\nthrough a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8,\nand HERC5, respectively. Ube2L6/UbcH8 plays a central role in ISGylation,\nunderscoring it as an important drug target for boosting innate antiviral\nimmunity. Depending on the type of conjugated protein and the ultimate\ntarget protein, E2 enzymes have been shown to function as monomers,\ndimers, or both. UbcH8 has been crystallized in both monomeric and\ndimeric forms, but its functional state remains unclear. Here, we\nused a combined approach of small-angle X-ray scattering (SAXS) and\nnuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s\noligomeric state in solution. SAXS revealed a dimeric UbcH8 structure\nthat could be dissociated when fused N-terminally to glutathione S-transferase.\nNMR spectroscopy validated the presence of a concentration-dependent\nmonomer–dimer equilibrium and suggested a back-side dimerization\ninterface. Chemical shift perturbation and peak intensity analysis\nfurther suggest dimer-induced conformational dynamics at the E1 and\nE3 interfaces, providing hypotheses for the protein’s functional\nmechanisms. Our study highlights the power of combining NMR and SAXS\ntechniques to provide structural information about proteins in solution.