Deubiquitinase A (DUBA) belongs to the ovarian tumor family of deubiquitinating enzymes and was initially identified as a negative regulator of type I interferons, whose overproduction has been linked to autoimmune diseases. The deubiquitinating activity of DUBA is positively regulated by phosphorylation at a single serine residue, S177, which results in minimal structural changes. We have previously shown that phosphorylation induces a two-state conformational equilibrium observed only in the active form of DUBA, highlighting the functional importance of DUBA dynamics. Here, we report the conformational dynamics of DUBA on the microsecond-to-millisecond time scales characterized by nuclear magnetic resonance relaxation dispersion experiments. We found that motions on these time scales are highly synchronized in the phosphorylated and nonphosphorylated DUBA. Despite the overall similarity of these two forms, different dynamic properties were observed in helix α1 and the neighboring regions, including residue S177, which likely contribute to the activation of DUBA by phosphorylation. Moreover, our data suggest that transient unfolding of helix α6 drives the global conformational process and that mutations can be introduced to modulate this process, which provides a basis for future studies to define the exact functional roles of motions in DUBA activation and substrate specificity.

Deubiquitinases deconjugate ubiquitin modifications from target proteins and are involved in many cellular processes in eukaryotes. The functions of deubiquitinases are regulated by post-translational modifications, mainly phosphorylation and ubiquitination. Post-translational modifications can result in subtle changes in structural and dynamic properties, which are difficult to identify but functionally important. In this work, we used NMR spectroscopy to characterize the conformational properties of the human deubiquitinase A (DUBA), a negative regulator of type I interferon. DUBA activity is regulated by phosphorylation at a single serine residue, Ser-177. We found that the catalytic rate constant of DUBA is enhanced by phosphorylation. By comparing NMR and enzyme kinetics data among different forms of DUBA with low and high activities, we concluded that a two-state equilibrium that was present only in phosphorylated DUBA is important for DUBA activity. Our results highlight the importance of defining conformational dynamics in understanding the mechanism of DUBA activation.

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Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies of Vibrio cholerae Pth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme.

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