Studying protein ubiquitination is difficult due to the innate complexity of the E1-E2-E3 ubiquitination cascade. Here we report the discovery that ubiquitin chemical probes can undergo a direct transthiolation reaction with the catalytic cysteine of the model HECT E3 ubiquitin ligase Rsp5 and form a catalytically active Rsp5~Ub thioester, in the absence of E1, E2 enzymes and ATP. The resulting Rsp5~Ub thioester undergoes efficient autoubiquitination, ubiquitinates protein substrates, and synthesizes polyubiquitin chains with specific isopeptide linkages. We show that the newly discovered two-component ubiquitination reaction recapitulates the enzymatic mechanism of the native ubiquitination reaction. Importantly, the developed two-component ubiquitination reaction provides the first direct evidence that E2 enzymes are dispensable for the polyubiquitin chain formation by HECT E3s. We have found that two other E3 enzymes, Nedd4-1 and RBR E3 Parkin, were also active under the new reaction conditions. The reported findings provide general mechanistic insights on the protein ubiquitination, and offer new strategies for assay development to discover pharmacological modulators of E3 enzymes.

Protein ubiquitination and formation of polyubiquitin chains without ATP, E1, and E2 enzymes

248th National Meeting (2014)

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