Ubiquitin-conjugating enzyme E2 variant (UEV) 3 lactate dehydrogenase (UEVLD) is a nuclear/cytosolic protein that is projected to have a near identical UEV domain to the well-studied protein Tsg-101. Tsg-101 is a crucial component of the endosomal sorting complex required for transport (ESCRT)-I complex, involved in lysosomal degradation, and can also be hijacked by retroviruses including HIV-1. AlphaFold predicts the respective UEV domains of each protein to be nearly identical in structure, despite the sequences only being 56% identical and 67% similar. To confirm this, the UEV domain of UEVLD is being structurally characterized through pseudocontact shift (PCS) experiments via paramagnetic NMR using lanthanide DOTA tags, which is significantly faster than traditional structural methods that involve primarily residual dipolar coupling (RDC) experiments. The solution structure has been solved using both the PCS restraints, and more traditional residual dipolar coupling (RDC) and nuclear overhauser enhancement (NOE) restraints. The resulting structure of the UEV domain from UEVLD was found to nearly overlap the AlphaFold predicted model, and to be nearly identical to the UEV domain of Tsg101. However, functional comparisons have indicated poor overlap of binding partners between the UEV domains of UEVLD and Tsg101. Overall, we have solved the structure of the UEV domain of UEVLD, which serves as a reference model to better understand the UEV domain of Tsg101, and provides a test case for structure determination by PCS restraints.
Vazquez et al. (2026) studied this question.