RESUMEN
Site-directed spin labeling (SDSL) has been invaluable in the analysis of protein structure and dynamics, and has been particularly useful in the study of membrane proteins. ExoU, an important virulence factor in Pseudomonas aeruginosa infections, is a bacterial phospholipase A2 that functions at the membrane - aqueous interface. Using SDSL methodology developed in the Hubbell lab, we find that the region surrounding the catalytic site of ExoU is buried within the tertiary structure of the protein in the soluble, apoenzyme state, but shows a significant increase in dynamics upon membrane binding and activation by ubiquitin. Continuous wave (CW) power saturation EPR studies show that the conserved serine hydrolase motif of ExoU localizes to the membrane surface in the active, holoenzyme state. SDSL studies on the C-terminal four-helix bundle (4HB) domain of ExoU similarly show a co-operative effect of ubiquitin binding and membrane association. CW power saturation studies of the 4HB domain indicate that two interhelical loops intercalate into the lipid bilayer upon formation of the holoenzyme state, anchoring ExoU at the membrane surface. Together these studies establish the orientation and localization of ExoU and the membrane surface, and illustrate the power of SDSL as applied to peripheral membrane proteins.
RESUMEN
Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange.
Asunto(s)
Células Endoteliales , Protones , Proteínas de Transporte de Anión/metabolismo , Células Endoteliales/metabolismo , Humanos , Ligandos , Lisofosfolípidos/metabolismo , Transducción de Señal , Esfingosina/metabolismoRESUMEN
Pseudomonas aeruginosa is an opportunistic multidrug-resistant pathogen and a common cause of infection in cystic fibrosis and ventilator-associated pneumonia and in burn and wound patients. P. aeruginosa uses its type III secretion system to secrete various effector proteins directly into mammalian host cells. ExoU is a potent type III secretion system effector that, after secretion, localizes to the inner cytoplasmic membrane of eukaryotic cells, where it exerts its phospholipase A2 activity upon interacting with ubiquitin and/or ubiquitinated proteins. In this study, we used site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the interaction of ExoU with soluble analogs of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). We found that dioctanoyl PI(4,5)P2 binds to and induces conformational changes in a C-terminal four-helix bundle (4HB) domain of ExoU implicated previously in membrane binding. Other soluble phosphoinositides also interacted with the 4HB but less effectively. Molecular modeling and ligand docking studies indicated the potential for numerous hydrogen bond interactions within and between interhelical loops of the 4HB and suggested several potential interaction sites for PI(4,5)P2 Site-directed mutagenesis experiments confirmed that the side chains of Gln-623 and Arg-661 play important roles in mediating PI(4,5)P2-induced conformational changes in ExoU. These results support a mechanism in which direct interactions with phosphatidylinositol-containing lipids play an essential role in targeting ExoU to host membrane bilayers. Molecules or peptides that block this interaction may prove useful in preventing the cytotoxic effects of ExoU to mitigate the virulence of P. aeruginosa strains that express this potent phospholipase toxin.