Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination

Conventional ubiquitination regulates key cellular processes by catalyzing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins(1). Recently, SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) of pathogenic Legionella pneumophila were shown to utilize NAD(+) to mediate phosphoribosyl-linked ubiquitination (PR-ubiquitination) of serine residues in host proteins(2,3). Yet, the molecular architecture of the catalytic platform enabling such a complex multistep process remained unknown. Here, we describe the structure of the catalytic core of SdeA composed of the mono-ADP-ribosyltransferase (mART) and the phosphodiesterase (PDE) domains and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that together with the mART-core creates a chamber for NAD(+) binding and ADP-ribosylation of Ub. The catalytic site in the PDE domain cleaves ADP-ribosylated Ub to phosphoribosyl Ub (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA:H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain juxtaposing the catalytic site that is essential for serine positioning for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination rather than modification of the cellular Ub pool determines the pathophysiological effect of SdeA during acute bacterial infection.