Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein
| Autor: | Ksenia Beyrakhova, Zhao-Qing Luo, Jianhua Zhao, Miroslaw Cygler, Yao Liu, Stephanie A. Bueler, John L. Rubinstein, Voula Kanelis, Michal T. Boniecki, Claudia P. Alvarez, Caishuang Xu, Li Xu |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
Adenosine Triphosphatase
0301 basic medicine Protein Conformation ATPase Plasma protein binding Pathology and Laboratory Medicine Biochemistry Legionella pneumophila Adenosine Triphosphate Protein structure Medicine and Health Sciences Macromolecular Structure Analysis Electron Microscopy lcsh:QH301-705.5 Microscopy Crystallography Effector Physics Condensed Matter Physics Bacterial Pathogens Enzymes 3. Good health Cell biology Legionella Pneumophila Chemistry Medical Microbiology Physical Sciences Crystal Structure Legionnaires' Disease Pathogens Protein Structure Determination Research Article lcsh:Immunologic diseases. Allergy Vacuolar Proton-Translocating ATPases Protein Structure Materials by Structure Chemical physics Protein subunit Materials Science Immunology Legionella Biology Research and Analysis Methods Crystals Microbiology Gene Expression Regulation Enzymologic 03 medical and health sciences Bacterial Proteins Virology Genetics Humans Solid State Physics Point Mutation V-ATPase Microbial Pathogens Molecular Biology Bacteria Organisms Phosphatases Biology and Life Sciences Proteins Electron Cryo-Microscopy Dimers (Chemical physics) biology.organism_classification Bacterial effector protein 030104 developmental biology lcsh:Biology (General) Mutation Enzymology biology.protein Parasitology lcsh:RC581-607 |
| Zdroj: | PLoS Pathogens, Vol 13, Iss 6, p e1006394 (2017) PLoS Pathogens |
| ISSN: | 1553-7374 1553-7366 |
| Popis: | Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires’ Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases) when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM) to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the ‘open’ conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK. Author summary V-ATPase-driven acidification of lysosomes in phagocytic cells activates enzymes important for killing of phagocytized pathogens. Successful pathogens can subvert host defenses by secreting effectors that target V-ATPases to inhibit lysosomal acidification or lysosomal fusion with other cell compartments. This study reveals the structure of the V-ATPase:SidK complex, an assembly formed from the interaction of host and pathogen proteins involved in the infection of phagocytic white blood cells by Legionella pneumophila. The structure and activity of the V-ATPase is altered upon SidK binding, providing insight into the infection strategy used by L. pneumophila and possibly other intravacuolar pathogens. |
| Databáze: | OpenAIRE |
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