Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.
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. pneumoph...
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doaj-25983daec897429b9ce82b455086e88c2020-11-25T02:02:15ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742017-06-01136e100639410.1371/journal.ppat.1006394Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.Jianhua ZhaoKsenia BeyrakhovaYao LiuClaudia P AlvarezStephanie A BuelerLi XuCaishuang XuMichal T BonieckiVoula KanelisZhao-Qing LuoMiroslaw CyglerJohn L RubinsteinIntracellular 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.http://europepmc.org/articles/PMC5469503?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jianhua Zhao Ksenia Beyrakhova Yao Liu Claudia P Alvarez Stephanie A Bueler Li Xu Caishuang Xu Michal T Boniecki Voula Kanelis Zhao-Qing Luo Miroslaw Cygler John L Rubinstein |
spellingShingle |
Jianhua Zhao Ksenia Beyrakhova Yao Liu Claudia P Alvarez Stephanie A Bueler Li Xu Caishuang Xu Michal T Boniecki Voula Kanelis Zhao-Qing Luo Miroslaw Cygler John L Rubinstein Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. PLoS Pathogens |
author_facet |
Jianhua Zhao Ksenia Beyrakhova Yao Liu Claudia P Alvarez Stephanie A Bueler Li Xu Caishuang Xu Michal T Boniecki Voula Kanelis Zhao-Qing Luo Miroslaw Cygler John L Rubinstein |
author_sort |
Jianhua Zhao |
title |
Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. |
title_short |
Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. |
title_full |
Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. |
title_fullStr |
Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. |
title_full_unstemmed |
Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. |
title_sort |
molecular basis for the binding and modulation of v-atpase by a bacterial effector protein. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS Pathogens |
issn |
1553-7366 1553-7374 |
publishDate |
2017-06-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC5469503?pdf=render |
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