E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and
regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub)
ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates.
The catalytic domain of HECT Ub ligases has a bilobal architecture that
separates the E2 binding region and catalytic site. An important question
regarding HECT domain function is the control of ligase activity and
specificity. Here we present a functional analysis of the HECT domain of the E3
ligase HUWE1 based on crystal structures and show that a single N-terminal helix
significantly stabilizes the HECT domain. We observe that this element modulates
HECT domain activity, as measured by self-ubiquitination induced in the absence
of this helix, as distinct from its effects on Ub conjugation of substrate
Mcl-1. Such subtle changes to the protein may be at the heart of the vast
spectrum of substrate specificities displayed by HECT domain E3 ligases.
