Abstract
Ubiquitination is important for numerous cellular processes in most eukaryotic organisms, including cellular proliferation, development, and protein turnover by the proteasome. The intestinal parasite Entamoeba histolytica harbors an extensive ubiquitin-proteasome system. Proteasome inhibitors are known to impair parasite proliferation and encystation, suggesting the ubiquitin-proteasome pathway as a viable therapeutic target. However, no functional studies of the E. histolytica ubiquitination enzymes have yet emerged. Here, we have cloned and characterized multiple E. histolytica ubiquitination components, spanning ubiquitin and its activating (E1), conjugating (E2), and ligating (E3) enzymes. Crystal structures of EhUbiquitin reveal a clustering of unique residues on the α1 helix surface, including an eighth surface lysine not found in other organisms, which may allow for a unique polyubiquitin linkage in E. histolytica. EhUbiquitin is activated by and forms a thioester bond with EhUba1 (E1) in vitro, in an ATP- and magnesium-dependent fashion. EhUba1 exhibits a greater maximal initial velocity of pyrophosphate:ATP exchange than its human homolog, suggesting different kinetics of ubiquitin activation in E. histolytica. EhUba1 engages the E2 enzyme EhUbc5 through its ubiquitin-fold domain to transfer the EhUbiquitin thioester. However, EhUbc5 has a >10-fold preference for EhUba1∼Ub compared with unconjugated EhUba1. A crystal structure of EhUbc5 allowed prediction of a noncovalent "backside" interaction with EhUbiquitin and E3 enzymes. EhUbc5 selectively engages EhRING1 (E3) to the exclusion of two HECT family E3 ligases, and mutagenesis indicates a conserved mode of E2/RING-E3 interaction in E. histolytica.