Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

Ender Volkan; Bradley A Ford; Jerome S Pinkner; Karen W Dodson; Nadine S Henderson; David G Thanassi; Gabriel Waksman; Scott J Hultgren

doi:10.1073/pnas.1207085109

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Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

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Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

Ender Volkan, Bradley A Ford, Jerome S Pinkner, Karen W Dodson, Nadine S Henderson, David G Thanassi, Gabriel Waksman and Scott J Hultgren

Proceedings of the National Academy of Sciences - PNAS, Vol.109(24), pp.9563-9568

06/12/2012

DOI: 10.1073/pnas.1207085109

PMCID: PMC3386062

PMID: 22645361

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Abstract

P pili are prototypical chaperone–usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone–subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone–adhesin-bound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone–adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone–subunit complexes except for the chaperone–terminator subunit (PapDH) and has a catalytic role in dissociating the NTD–PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTD–Plug complex bound all of the chaperone–subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone–subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone–usher pathway pilus.

macromolecular assembly

Biological Sciences

virulence factor

bacterial pathogenesis

biolayer interferometry

urinary tract infections

Details

Title: Subtitle: Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine
Creators: Ender Volkan - Department of Molecular Microbiology
Bradley A Ford - Center for Women’s Infectious Diseases Research
Jerome S Pinkner - Department of Molecular Microbiology
Karen W Dodson - Department of Molecular Microbiology
Nadine S Henderson - Center for Infectious Diseases, Department of Molecular Genetics and Microbiology
David G Thanassi - Center for Infectious Diseases, Department of Molecular Genetics and Microbiology
Gabriel Waksman - Institute of Structural and Molecular Biology
Scott J Hultgren - Department of Molecular Microbiology
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.109(24), pp.9563-9568
DOI: 10.1073/pnas.1207085109
PMID: 22645361
PMCID: PMC3386062
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences
Alternative title: Mechanism of action of PapC in pilus assembly
Language: English
Date published: 06/12/2012
Academic Unit: Pathology
Record Identifier: 9984047683002771

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