Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex

L. M. Sampaleanu, Jeffrey B. Bonanno, M. Ayers, J. Koo, S. Tammam, S. K. Burley, Steven C. Almo, L. L. Burrows, P. L. Howell

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 Å resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

Original languageEnglish (US)
Pages (from-to)143-159
Number of pages17
JournalJournal of Molecular Biology
Volume394
Issue number1
DOIs
StatePublished - Nov 20 2009

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Pseudomonas aeruginosa
Membranes
Bacterial Fimbriae
Ferredoxins
Proteins
Virulence Factors
Molecular Biology
Type II Secretion Systems

Keywords

  • PilN
  • PilO structure
  • Pseudomonas aeruginosa
  • Type II secretion
  • type IV pili

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex. / Sampaleanu, L. M.; Bonanno, Jeffrey B.; Ayers, M.; Koo, J.; Tammam, S.; Burley, S. K.; Almo, Steven C.; Burrows, L. L.; Howell, P. L.

In: Journal of Molecular Biology, Vol. 394, No. 1, 20.11.2009, p. 143-159.

Research output: Contribution to journalArticle

Sampaleanu, L. M. ; Bonanno, Jeffrey B. ; Ayers, M. ; Koo, J. ; Tammam, S. ; Burley, S. K. ; Almo, Steven C. ; Burrows, L. L. ; Howell, P. L. / Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex. In: Journal of Molecular Biology. 2009 ; Vol. 394, No. 1. pp. 143-159.
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AU - Tammam, S.

AU - Burley, S. K.

AU - Almo, Steven C.

AU - Burrows, L. L.

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AB - Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 Å resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

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