STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF UNCONVENTIONAL BACTERIAL UBIQUITINATION

Abstract

Legionella pneumophila, a gram-negative intracellular pathogen, is the causative agent of a severe form of pneumonia termed Legionnaires’ disease. Following engulfment by alveolar macrophages, Legionella secretes ~330 effector proteins into the host cell. These proteins facilitate the formation of the Legionella-Containing Vacuole (LCV), a replicative niche that allows Legionella to escape phagosomal degradation and subsequently permits intracellular bacterial replication. A growing number of effectors have been reported to alter host ubiquitin signaling, and further characterization of these effectors is crucial for understanding how host defenses are eluded. Here, I present work that identifies the underlying mechanisms that govern E2 binding and ubiquitin transfer to a Legionella-encoded E3 ligase SidC. We have shown that the insertion (INS) domain of SidC is important for binding of ubiquitin-loaded E2. To define the key residues critical for this interaction, we solved the crystal structure of the SidC paralog SdcA in complex with an E2. We also crystallized and solved the structure of the catalytic domain of SidC in complex with its preferred E2 covalently conjugated to ubiquitin. This ternary structure reveals the molecular interactions that lead to the transfer of ubiquitin from E2 to SidC. This study furthers our understanding of the mechanism by which this novel E3 ligase carries out its function. The SidE family of effectors catalyzes a form of ubiquitination that functions independently of the canonical E1, E2, and E3 enzymatic cascade. However, the mechanism by which this reaction is carried out remained elusive. Here, we present the crystal structure of a fragment of the SidE family member SdeA that retains full ubiquitination activity. Our structure reveals that the ubiquitination catalytic module contains two distinct domains, a phosphodiesterase domain (PDE) and a mono-ADP-ribosyltransferase (mART) domain. Biochemical analysis shows that the conversion of ubiquitin to ADP-ribose-ubiquitin (ADPR-Ub) and the ligation of ADPR-Ub to substrates are two independent activities of SdeA. Furthermore, our crystal structures of a homologous PDE domain from the L. pneumophila effector SdeD in complex with both Ub and ADPR-Ub reveals an intriguing mechanism of how SdeA processes ADPR-Ub to release AMP and conjugates PR-Ub to serine residues of substrates.