Structural Basis For Rabl Recognition By Legionella Pneumophila Effector LidA

The intracellular pathogen Legionella pneumophila infects into human alveolar macrophages, delivers275validated substrates into the host cytosol by its Dot/Icm type IV secretion system. It hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication.Maturation of the LCV involved acquisition of Rabl, a key regulator involved in the host ER-Golgi vesicular trafficking. The recruitment of Rabl is mediated by the Dot/Icm effector protein SidM. SidM is a bifunctional enzyme having the activity of both Rabl-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). The mechanism of SidM-induced Rabl recruitment has been discovered by now.LidA, another Rabl-interacting Dot/Icm effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rabl to the LCV as well. LidA has the auxiliary activity for Rab1recruitment, whereas it is more important for the formation of the replication vacuole compared with SidM/DrrA by knockout experiments. To reveal how LidA recognize the host Rab1, we expressed and purified the complexes of LidA core coiled-coil domain with both GDP-bound and GTP-bound Rabl protein, and solved the crystal structures of LidA coiled-coil domain in complex with two different states of Rab1, GDP-and GTP-bound, by co-crystallization and X-ray diffraction experiments.The overall LidA-Rab1complex adopts a compact and globular structure. The interaction between LidA and Rab1resulted in a1:1stoichiometric complex and showed exceptionally compact binding manner. In the complex, LidA (residues224-559) arrange into a structure resembling a hand with the baby finger buried in the palm and the remaining four straightened composed of seven a-helices and ten β-strands, Six of the seven a-helices form three extended two-stranded a-helical coiled-coils, along with the islated a6and the following (3-sheets, form into four fingers of LidA. Rabl is held in a large and pronounced groove made by the four "fingers" with an extensive, though not complete, charge and surface complementarities, and buried41%in the structure. Contacts between LidA and Rabl are established through a combination of hydrogen bonds and hydrophobic interactions involving the switch Ⅰ, switch Ⅱ and the interswitch region of Rabl.Surprisingly, Structural comparison revealed that GDP-Rab1bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rabl and render it persistently active. As with GTP, LidA maintains GDP-Rab1in the active conformation through interaction with its two conserved switch regions by its C-terminal region. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP-and GTP-Rab1equally well with an affinity approximately7.5nM, and identified the LidA C-terminal function. We also mapped the minimal interaction region of LidA for recognizing Rabl by domain ampping; found LidA can separate SidM-Rabl complex and bind with Rab1competively by gel filtration chromatography; cofirmed the interaction between LidA and AMP-Rab1by mass method and ITC; moreover, we found LidA not only recognize Rab1,Rab6,Rab8as reported before, but also form complexes with Rab2,Rab,Rab5,Rab7,Rab9,Rabl1,Rab14,Rab18,Rab20and Rab22。Taken together, our results reveal the LidA can recognize Rab1by the special hand-like conformation in a high affinity, and support a unique mechanism by which a bacterial effector protein regulates Rabl recycling. Our results add a new insight into the regulation of the host Rabl membrane cycle by pathogen-secreted coiled-coil effector.