Ubiquitin chain conformation regulates recognition and activity of interacting proteins

Abstract

Mechanisms of protein recognition have been extensively studied for single-domain proteins ¹ , but are less well characterized for dynamic multi-domain systems. Ubiquitin (Ub) chains represent a biologically important multi-domain system that requires recognition by structurally diverse Ub-interacting proteins (UbIPs) ^{2, 3} . Ub chain conformations in isolation are often different from conformations observed in UbIP complexes, suggesting either great dynamic flexibility or extensive chain remodeling upon binding.

Using single-molecule FRET, we show here that Lys63-, Lys48- and Met1-linked diUb exist in several distinct conformational states in solution. Lys63- and Met1-linked diUb adopt extended ‘open’ and more compact ‘closed’ conformations, and Ub binding domains (UBDs) and deubiquitinases (DUBs) select pre-existing conformations. In contrast, Lys48-linked diUb adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel Ub chains to hydrolyze the isopeptide bond. Disruption of the Lys48-diUb interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in Ub chains provide an additional layer of regulation in the Ub system, and distinct conformations observed in differently linked polyUb may contribute to the specificity of UbIPs.