Author response: Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E. coli F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk’s ε subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides.

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Faculty Opinions recommendation of Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727132410.793528380 ◽

2017 ◽

Author(s):

Dario Leister ◽

Thilo Rühle

Keyword(s):

Atp Synthase ◽

Rotational States ◽

E Coli

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Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

Applied and Environmental Microbiology ◽

10.1128/aem.01704-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 6953-6963 ◽

Cited By ~ 5

Author(s):

Zhe Zhao ◽

Lauren J. Eberhart ◽

Lisa H. Orfe ◽

Shao-Yeh Lu ◽

Thomas E. Besser ◽

...

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Disulfide Bonds ◽

Gene Knockout ◽

Single Gene ◽

Site Directed Mutagenesis ◽

Content Type ◽

E Coli ◽

Synthase Inhibitor ◽

Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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Author response for "A rational approach to improving titer in E. coli ‐based cell‐free protein synthesis reactions"

10.1002/btpr.3062/v2/response1 ◽

2020 ◽

Author(s):

Noelle Colant ◽

Beatrice Melinek ◽

Jaime Teneb ◽

Stephen Goldrick ◽

William Rosenberg ◽

...

Keyword(s):

Protein Synthesis ◽

Author Response ◽

Rational Approach ◽

Free Protein ◽

E Coli ◽

Synthesis Reactions ◽

Cell Free Protein Synthesis

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Structure of a bacterial ATP synthase

10.1101/463968 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L. Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

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The role of the amino‐terminal periplasmic domain of subunit a of the E. coli ATP synthase in function and assembly

The FASEB Journal ◽

10.1096/fasebj.20.4.a518-d ◽

2006 ◽

Vol 20 (4) ◽

Author(s):

Steven B Vik ◽

Robert R Ishmukhametov

Keyword(s):

Atp Synthase ◽

Subunit A ◽

E Coli ◽

Amino Terminal ◽

Periplasmic Domain

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Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽

10.3390/antibiotics10121456 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1456

Author(s):

Amaravadhi Harikishore ◽

Chui-Fann Wong ◽

Priya Ragunathan ◽

Dennis Litty ◽

Volker Müller ◽

...

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Membrane Vesicles ◽

Α Subunit ◽

Rotational States ◽

C Terminus ◽

Inverted Membrane Vesicles ◽

Hydrolysis Of ◽

Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

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