Solution NMR structure and NMR-based molecular backbone dynamics of Ub^KEKS: a ubiquitin variant encoded in the UBB4...

Recently, proteomics analyses using databases of unannotated ORFs revealed that ubiquitin (Ub) variants can be encoded and expressed from pseudogenes. One such pseudogene, UBBP4, produces Ub^KEKS, which contains four substitutions (Q2K, K33E, Q49K, and N60S) relative to canonical Ub. Unlike Ub, Ub^KEKS does not promote proteasomal degradation through K48 linkages and instead modifies a distinct set of proteins. To elucidate the structural basis of this divergence, we solved the NMR solution structure of Ub^KEKS and characterized its backbone dynamics by 15N-relaxation. While Ub^KEKS retains the overall helix-grip fold, we observed significant rearrangements and amplified motions in residues governing the Ub pincer mode, a conformational switch that determines whether UIMs engage the canonical I44 interface or the α1-β3 edge. Specifically, Q2K and K33E cooperate to enhance motions on both fast (ps-ns) and slow (µs-ms) timescales within α1, the β1-β2 loop, and β5-regions central to pincer mode regulation. In addition, Q49K, adjacent to I44, perturbs UIM recognition and likely interferes with K48 chain formation and binding to the proteasomal receptor S5a. Collectively, our findings identify structural and dynamical determinants that explain Ub^KEKS's distinct substrate profile and inability to target proteins for degradation.