A Legionella Effector Sidc – A New Family Of E3 Ubiquitin Ligase With A Specific Pi(4)P Binding Domain

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The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication permissive compartment known as the Legionella-containing vacuole (LCV). Among these proteins, SidC anchors to the cytoplasmic surface of the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] and is important for the recruitment of host endoplasmic reticulum (ER) proteins to this organelle. However, the biochemical function underlying this activity is unknown. We first determined the structure of the N-terminal domain of SidC, which has no structural homology to any protein. Sequence homology analysis revealed a potential canonical catalytic triad formed by Cys46, His444, and Asp446 on the surface of SidC. Unexpectedly, we found that SidC is an E3 ubiquitin ligase which utilizes the C-H-D triad to catalyze the formation of high molecular weight poly-ubiquitin chains through multiple ubiquitin lysine residues. A C46A mutation completely abolished the E3 ligase activity and the ability of the protein to recruit host ER proteins as well as poly-ubiquitin conjugates to the LCV. Thus, SidC represents a novel E3 ubiquitin ligase family important for phagosomal membrane remodeling by L. pneumophila. Next, we also reported the crystal structure of SidC (1-871). The structure revealed that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) is comprised of a four !-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we further found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC to the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical for its function in the remodeling of bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows potential to be developed as a sensitive and accurate PI(4)P probe in living cells.

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SidC; E3 Ubiquitin Ligase; PI(4)P


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Union Local


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Feigenson,Gerald W

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Ph. D., Biophysics

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Doctor of Philosophy

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dissertation or thesis

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