Functional Dynamics of Deubiquitinase A.

Deubiquitinase A (DUBA) is an isopeptidase that cleaves K63-linked and K48-linked ubiquitin chains from substrate proteins. DUBA belongs to the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs) and was initially identified as a negative regulator of the production of type I interferons. DUBA is also known to regulate the production of several other cytokines in both innate and adaptive immune cells. More recently, its role in regulation of the stability of chromatin regulators essential for human development has been revealed. DUBA is a model system for understanding how the activity and substrate specificity of DUBs are regulated by post-translation modifications (PTMs) through modulation of the conformational ensemble that pre-exists in the apo form of the enzyme. We have previously identified changes in the conformational ensemble of DUBA upon phosphorylation, which is essential for DUBA activity, using solution nuclear magnetic resonance (NMR) spectroscopy and activity assays. We have also characterized the conformational dynamics on the microsecond-to-millisecond timescales using NMR relaxation dispersion experiments. We will present NMR and activity data on several mutants of DUBA, which allowed us to identify the structural determinants of the previously observed conformational dynamics and to understand the functional roles of motions in DUBA. We will also present NMR data on the enzyme-substrate complex, which shed light on the mechanism for the specificity of DUBA for the K63-linked ubiquitin chain. Overall, our data suggest that the conformational dynamics of DUBA can be modulated by phosphorylation and are essential for the function of DUBA. Our study highlights the functional importance of conformational dynamics in the DUB family and the need to define dynamic properties of DUBs in order to fully understand how activity and substrate specificity can both be modulated by PTMs.