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Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

Significance A long-standing question in the field of microbial pathogenesis is how virulence factors are regulated within host cells and how their activity is specifically directed toward a particular host cell compartment. Legionella pneumophila resolves this dilemma by tightly coupling the phospholipase A1 activity of one of its effectors, vacuolar protein sorting inhibitor protein D (VipD), to this protein’s interaction with endosomal host GTPases. We now present the crystal structure of VipD in complex with host cell Rab5c, providing a detailed look into the ingenious molecular mechanisms underlying the allosteric activation of a virulence factor by a host protein and its spatiotemporal regulation. These results open the path for the development of novel therapeutics aimed at blocking the VipD activation process rather than the enzyme’s active site. A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.