Abstract: AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus
far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also
encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle
transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like
domain of SidD. Here, we determined the crystal structure of full length SidD including the
uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within
the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus
within mammalian cells. Deletion of the loop (??loop) or substitution of its aromatic phenylalanine
residues rendered SidD cytosolic, showing that the hydrophobic loop is the
primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal
alpha helices resulted in a CTD variant incapable of discriminating between membranes of
different composition. Moreover, a L. pneumophila strain producing SidD??loop phenocopied
a L. pneumophila ??sidD strain during growth in mouse macrophages and displayed prolonged
co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting
of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation
during L. pneumophila infection.
