Insights into catalysis and function of phosphoribosyl-linked serine ubiquitination

Abstract

Conventional ubiquitination regulates key cellular processes by catalyzing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins1. 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 proteins2,3. 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.

Targeting ADP-ribosylation as an antimicrobial strategy

“…SdeA catalytic mechanism is inhibited by adenosine 5′-O-thiomonophosphate, an AMP analogue, that binds with low-affinity the SdeA PDE domain and alters substrate positioning. This finding provides new insights for a rationale designing of novel antimicrobial compounds to inhibit this class of Legionella toxins [248].…”

“…By clarifying the virulence mechanism of SdeA, it has been shown that the ubiquitination induced by Legionella in the host is dependent on ADPr [245]. Structural and biochemical characterisation have revealed that SdeA is a dual enzyme with both MARylation and phosphodiesterase (PDE) activities, due to the presence of two distinct protein folds, namely ARTC-like and PDE domains [245,247,248]. ADPr-dependent ubiquitination of protein substrates catalysed by SdeA is a two-step reaction: first, SdeA transfers ADP-ribose from NAD + onto arginine 42 (Arg42) of an Ubiquitin (Ub) molecule in order to generate an ADP-ribosylated-Ub intermediate; in the second step, phosphodiesterase activity converts ADP-ribosylated-Ub intermediate to phosphoribosyl-Ub which is then conjugated through an ester linkage to a serine residue to target protein or to SdeA itself [245,248].…”

Deciphering the catalytic mechanism of bacterial ubiquitination

“…Magnetic forms of graphene would be useful for spintronics, a technology that forms the basis of today's magnetic data storage 3,4 . But the main interest in generating magnetic edge states in graphene is for quantum technologies.…”

“…Once the nicotinamide group from NADH is released from the enzyme, a conformational change can occur, allowing Arg42 to replace Arg72 in the active site. This model explains why ADPR attaches selectively to Arg42 and not to , Dong et al 2 and Kalayil et al 3 report the structure of the enzyme SdeA, and Wang et al 4 present the structure of the enzyme SidE. a, In the first step, the enzyme's mART domain processes NAD + and adds an adenosine diphosphate ribose (ADPR) group to the amino-acid residue arginine 42 (Arg42) of ubiquitin.…”

“…The four papers [1][2][3][4] provide a detailed picture of the bacterial reaction pathway, with complementary insights into the catalytic mechanisms. Akturk, Dong and Kalayil, and their respective colleagues, report atomic structures of SdeA's catalytic core, which consists of the PDE and mART domains.…”