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

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.Microbial pathogens have evolved numerous ways to subvert and exploit normal host cell processes and to cause disease. Intravacuolar pathogens use specialized translocation devices such as type IV secretion systems (T4SS) to deliver virulence proteins, so-called effectors, across the bacterial and host cell membrane into the cytosol of the infected cell (1–3). Many of the translocated effectors studied to date alter cellular events such as vesicle trafficking, apoptosis, autophagy, protein ubiquitylation, or protein synthesis, among others, thereby creating conditions that support intracellular survival and replication of the microbe (4, 5). Bacteria with a nonfunctional T4SS are often avirulent and degraded along the endolysosomal pathway, thus underscoring the importance of translocated effectors for microbial pathogenesis.Although T4SS-mediated effector translocation may be a convenient way for pathogens to manipulate host cells from within the safety of their membrane-enclosed compartment, it also creates a challenging dilemma: how can the bacteria ensure that their translocated effectors reach the correct host cell target for manipulation, and how can they prevent them from indiscriminately affecting bystander organelles or proteins that may otherwise be beneficial for intracellular survival and replication of the microbe? It is reasonable to expect that regulatory mechanisms have evolved that restrain the catalytic activity of effectors. Although detailed insight into these processes is scarce, an emerging theme among effectors is that their enzymatic activity is functionally coupled to their interaction with a particular host factor. For example, SseJ from Salmonella enterica serovar Typhimurium displays glycerophospholipid-cholesterol acyltransferase activity only on binding to the active GTPases RhoA, RhoB, or RhoC (6–8). Likewise, Pseudomonas aeruginosa ExoU requires mono- or poly-ubiquitinated proteins for the activation of its phospholipase A2 (PLA₂) domain (9), whereas Yersinia YpkA exhibits kinase activity only in the presence of host cell actin (10). Exactly how binding to host ligands results in the activation of these translocated effectors remains unclear because no structural information for these protein complexes is available.VipD is a T4SS-translocated substrate of Legionella pneumophila, the causative agent of a potentially fatal pneumonia known as Legionnaires'' disease, and another example of an effector whose catalytic activity depends on the presence of a host factor (11–14). Following uptake by human alveolar macrophages, L. pneumophila translocates VipD together with more than 250 other effector proteins through its Dot/Icm T4SS into the host cell cytoplasm (15). These effectors act on numerous host processes to mediate evasion of the endolysosomal compartment and to establish a Legionella-containing vacuole (LCV) that supports bacterial growth (16). Although the precise biological role of most L. pneumophila effectors remains unclear, we recently showed that VipD is important for endosomal avoidance by LCVs. The protein localizes to endosomes presumably by binding to the small GTPases Rab5 or Rab22, key regulators of endosomal function (13, 14). Rab GTPase binding to the C-terminal domain of VipD triggers robust phospholipase A1 (PLA₁) activity within the N-terminal domain, resulting in the removal of phosphatidylinositol 3-phosphate PI(3)P] and potentially other lipids from endosomal membranes (14). Without PI(3)P, endosomal markers such as early endosomal antigen 1 (EEA1) are lost from these membranes, most likely rendering the endosomal compartment fusion incompetent (17). L. pneumophila mutants lacking vipD are attenuated in avoiding endosomal fusion, and their LCVs acquire the endosomal marker Rab5 more frequently than LCVs containing the parental strain producing VipD (14). Thus, by coupling PLA₁ activity to Rab5 binding, the catalytic activity of VipD is directed specifically against the endosomal compartment without visibly affecting neighboring cell organelles.VipD was originally identified in a screen for L. pneumophila effectors that interfere with the vacuolar sorting pathway in yeast (11). The N-terminal half of VipD possesses high homology to patatin, a lipid acyl hydrolase present in the potato tuber (12, 13). Analogous to other patatin-like proteins, VipD harbors a conserved serine lipase motif Gly-x-Ser-x-Gly (x = any amino acid) as part of a Ser-Asp catalytic dyad that, together with two consecutive glycine residues (Asp-Gly-Gly motif), is expected to stabilize the oxyanion intermediate during the acyl chain cleavage (13). Mutation of these conserved catalytic residues in VipD results in loss of PLA₁ activity (14), confirming their role in substrate hydrolysis.The recently reported crystal structure of VipD confirmed the predicted bimodular organization (13) and, in addition, revealed a surface loop, called “lid” in other phospholipases, that shields the entry to the catalytic site. The inhibitory lid may explain why purified recombinant VipD alone exhibits little or no PLA₁ activity in vitro. However, given that binding of Rab5 or Rab22 to VipD activates the PLA₁ activity within the N-terminal region (14), we wondered if and how this binding event causes the inhibitory lid to be removed to render the active site substrate accessible.Using an integrative approach involving X-ray crystallography, molecular dynamics, biochemistry, and cellular imaging, we now deciphered at a molecular level the mechanism that stimulates the intrinsic PLA₁ activity of VipD and determined the underlying specificity for the VipD–Rab5 interaction and endosomal targeting.