Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion

Abstract

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin signaling pathways via installing mono-ADP-ribosylation on threonine 66 of ubiquitin. However, the molecular basis of substrate recognition by CteC is not well-understood. In this paper, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between ubiquitin and structurally close ubiquitin-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.