Solution NMR Structure and NMR-based Molecular Backbone Dynamics of UbKEKS: A Ubiquitin Variant Encoded in the UBB4 Pseudogene with Emerging Specific and New Functions.

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 UbKEKS, which contains four substitutions (Q2K, K33E, Q49K, N60S) relative to canonical Ub. Unlike Ub, UbKEKS 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 UbKEKS and characterized its backbone dynamics by 15N-relaxation. While UbKEKS 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 UbKEKS's distinct substrate profile and inability to target proteins for degradation.