Reprogramming a Deubiquitinase into a Transamidase

Access
to well-defined ubiquitin conjugates has been key to elucidating the
biochemical functions of proteins in the ubiquitin signaling network.
Yet, we have a poor understanding of how deubiquitinases and ubiquitin-binding
proteins respond to ubiquitin modifications when anchored to a protein
other than ubiquitin or a ubiquitin-like protein. This is due to the
difficulty of synthesizing ubiquitinated proteins comprised of native
isopeptide bonds. Here we report on the evolution of a deubiquitinase
capable of site-specifically modifying itself with defined ubiquitin
chains. Following mutagenesis and yeast display screening, we identify
a variant of the yeast ubiquitin C-terminal hydrolase Yuh1 that has
a 28-fold improvement in the transamidation to hydrolysis ratio relative
to the wild type enzyme. The switch in activity enables robust autoubiquitination
of a lysine in the crossover loop to form an isopeptide bond. We demonstrate
the utility of autoubiquitinating the evolved Yuh1 variant by investigating
the consequences of ubiquitin chain anchoring on the activities of
other deubiquitinases. Much to our surprise, we find that certain
deubiquitinases are exquisitely sensitive to chain anchoring. These
results highlight the importance of investigating the biochemical
activities of deubiquitinases with both substrate-anchored and unanchored
ubiquitin chains.