Role of Dimerization in Poly-Ubiquitin Chain Formation

Abstract

The ubiquitin proteolysis pathway is responsible for protein degradation utilizing three enzymes, the ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2), and a ligating enzyme (E3), that respectively activate, transfer and ligate ubiquitin (Ub) onto a target protein. Repeated cycles of this process results in a poly-ubiquitinated target protein that is degraded by the 26S proteasome. How the poly-Ub chains are formed remains unknown. One suggested model involves the dimerization of an E2 enzyme allowing Ub passage and ligation between adjacent E2 enzymes. We are examining this mechanism for the E2 enzymes Ubc1 and HIP2, which contain C-terminal UBA domains that allows for non-covalent Ub binding in addition to a thioester-bound Ub. The dimerization of these E2 enzymes was tested using sedimentation equilibrium and small angle x-ray scattering and showed that both are monomeric. Disulphide-bound E2-Ub complexes were used to mimic the thiolester, and these complexes had a weak propensity to dimerize. This was supported in ubiquitination assays that showed a thioester-bound Ub on an E2 could be transferred to a non-hydrolyzable, disulphide-bound Ub molecule on a second E2 enzyme. This suggests weak dimerization is likely sufficient to allow the first step of poly-Ub chain formation. We tested whether the length of the poly-Ub chains on HIP2 stimulates dimerization by creating HIP2-Ub2 and HIP2-Ub4 complexes. The ability of these species to dimerize was assessed via sedimentation equilibrium and NMR spectroscopy. The isolated UBA domain from HIP2 was used in competition experiments to determine how it might influence poly-Ub chain formation. This work provides the first structural evidence for poly-Ub chain formation as assembled on the E2 enzyme.