Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport

Simpson, Brent W.; Pahil, Karanbir S.; Owens, Tristan W.; Lundstedt, Emily A.; Davis, Rebecca M.; Kahne, Daniel; Ruiz, Natividad

doi:10.1128/mbio.01931-19

Title: Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport

Journal Article · Tue Aug 20 00:00:00 EDT 2019 · mBio (Online)

DOI:https://doi.org/10.1128/mbio.01931-19· OSTI ID:1569912

Simpson, Brent W. ^[1]; Pahil, Karanbir S. ^[2]; Owens, Tristan W. ^[2]; Lundstedt, Emily A. ^[3]; Davis, Rebecca M. ^[3]; Kahne, Daniel ^[4];

^[3]

The Ohio State Univ., Columbus, OH (United States); Univ. of Georgia, Athens, GA (United States)
Harvard Univ., Cambridge, MA (United States)
The Ohio State Univ., Columbus, OH (United States)
Harvard Univ., Cambridge, MA (United States); Harvard Medical School, Boston, MA (United States)

ATP-binding cassette (ABC) transporters constitute a large family of proteins present in all domains of life. They are powered by dynamic ATPases that harness energy from binding and hydrolyzing ATP through a cycle that involves the closing and reopening of their two ATP-binding domains. The LptB₂FGC exporter is an essential ABC transporter that assembles lipopolysaccharides (LPS) on the surface of Gram-negative bacteria to form a permeability barrier against many antibiotics. LptB₂FGC extracts newly synthesized LPS molecules from the inner membrane and powers their transport across the periplasm and through the outer membrane. How LptB₂FGC functions remains poorly understood. Here, we show that the C-terminal domain of the dimeric LptB ATPase is essential for LPS transport in Escherichia coli. Specific changes in the C-terminal domain of LptB cause LPS transport defects that can be repaired by intragenic suppressors altering the ATP-binding domains. Surprisingly, we found that each of two lethal changes in the ATP-binding and C-terminal domains of LptB, when present in combined form, suppressed the defects associated with the other to restore LPS transport to wild-type levels both in vivo and in vitro. We present biochemical evidence explaining the effect that each of these mutations has on LptB function and how the observed cosuppression results from the opposing lethal effects these changes have on the dimerization state of the LptB ATPase. We therefore propose that these sites modulate the closing and reopening of the LptB dimer, providing insight into how the LptB2FGC transporter cycles to export LPS to the cell surface and how to inhibit this essential envelope biogenesis process.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC); National Institutes of Health (NIH)

Grant/Contract Number:: R01-GM100951; R01-GM066174; R0-AI081059

OSTI ID:: 1569912

Journal Information:: mBio (Online), Vol. 10, Issue 4; ISSN 2150-7511

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 11 works

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Title: Combining Mutations That Inhibit Two Distinct Steps of the ATP Hydrolysis Cycle Restores Wild-Type Function in the Lipopolysaccharide Transporter and Shows that ATP Binding Triggers Transport

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