Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1

Abstract

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Clickable report tags for identification of modified peptides by mass spectrometry

“…30 First, in an in vitro reaction of the well-described pair of AMP-transferase DrrA and its substrate Rab1b with ATP or N 6 -propargyl ATP the AMPylated Rab1b was prepared and characterized by intact protein MS (Figure S5 and S6). 31,32 Both, wt and N 6 -propargyl modified proteins were then coupled with DMP-tag, reduced, alkylated and trypsinized. The resulting peptide mixture was analyzed by direct injection into the mass spectrometer.…”

AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins

“…Therefore, we have tested the possibility to capture the monophosphate moiety of AMPylated proteins using an alkoxide-bridge Mn 2+ metal complex bound in the gel by Phos-tag ligand. We have used a recombinant Rab1b protein, which was AMPylated in vitro by the recombinant bacterial effector DrrA ( Du et al., 2021 ). The Rab1b identities were confirmed by top-down mass spectrometry ( Figure S4 ).…”

Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology

“…Cosubstrate mediated cross-linking not only helped to study substrate recognition of enzymes but also served to identify an unexpected enzymatic activation mechanism of the Legionella effector DrrA/SidM . As mentioned before, DrrA is a multidomain effector enzyme secreted to host cytosol during infection and interferes with membrane trafficking via its GEF and AMP transfer activities toward the human small GTPase Rab1. , While the concept of genetic code expansion allowed structural insights to be obtained on DrrA GEF activity by cross-linking DrrA GEF –Rab1 (as discussed in section ), the molecular details on AMP transfer by the ATase domain (DrrA ATase ) were incomplete .…”

“…As mentioned before, DrrA is a multidomain effector enzyme secreted to host cytosol during infection and interferes with membrane trafficking via its GEF and AMP transfer activities toward the human small GTPase Rab1. , While the concept of genetic code expansion allowed structural insights to be obtained on DrrA GEF activity by cross-linking DrrA GEF –Rab1 (as discussed in section ), the molecular details on AMP transfer by the ATase domain (DrrA ATase ) were incomplete . In a recent study, the covalently linked DrrA ATase –Rab1 complex was obtained which allowed successful structure determination by X-ray crystallography . Surprisingly, the complex structure revealed that Rab1 was unexpectedly cross-linked to the backside instead of the catalytic site of DrrA ATase .…”