Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP

Abstract

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

Novel Mechanism Coupling Cyclic AMP-Protein Kinase A Signaling and Golgi Trafficking via Gyp1 Phosphorylation in Polarized Growth

“…Our discovery of Gyp1 as a direct substrate of PKA reveals an important signaling node for this control. Interestingly, it has been reported that the bacterial pathogen Legionella pneumophila secrets proteins that mimic the activity of eukaryotic Rab1 GAP to manipulate the host secretory pathway (72,73). These findings suggest that Rab1/Ypt1 GAPs could be targets for effective regulation and manipulation of the eukaryotic secretory process throughout evolution.…”

Vibrio cholerae T3SS Effector VopE Modulates Mitochondrial Dynamics and Innate Immune Signaling by Targeting Miro GTPases

“…Our data shows that VopE exerts its activity through binding to GTPase-I of Miro, thus it is likely that the molecular mechanism of the VopE-Miro interaction is similar to that of Rho GTPase inhibition by Yersinia YopE, Pseudomonas aeruginosa ExoS, and Salmonella enterica SptP effectors (Stebbins and Galan, 2000). However, a crystal structure of VopE is required to confirm this hypothesis as the unique tertiary structures and catalytic mechanisms of some T3SS effectors such as LepB of Legionella, VirA of Shigella and EspG of EPEC were not apparent from their primary or predicted secondary structures (Dong et al, 2012; Yu et al, 2013). …”

Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

“…For example, LidA binds Rab1, which stabilizes its activation (46) and promotes the acquisition of ER-derived vesicles (47). Furthermore, DrrA/SidM is a GEF that posttranslationally modifies Rab1 with AMP (48-51), thereby preventing inactivation by GAP proteins (52)(53)(54). Rab1 (12), as well as the regulators of endocytic traffic Rab5 and Rab7, have been implicated in C. burnetii PV biogenesis (15,55,56).…”