Characterization of the PilN, PilO and PilP type IVa pilus subcomplex

Authors

  • S. Tammam,

    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
    2. Department of Biochemistry, Faculty of Medicine, University of Toronto, 1 King's Circle, Toronto, Ontario, M5S 1A8, Canada.
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  • L. M. Sampaleanu,

    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
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  • J. Koo,

    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
    2. Department of Biochemistry, Faculty of Medicine, University of Toronto, 1 King's Circle, Toronto, Ontario, M5S 1A8, Canada.
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  • P. Sundaram,

    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
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  • M. Ayers,

    1. Department of Biochemistry and Biomedical Sciences, and The Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main St. W., Hamilton, Ontario, L8N 3Z5, Canada
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  • P. Andrew Chong,

    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
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  • J. D. Forman-Kay,

    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
    2. Department of Biochemistry, Faculty of Medicine, University of Toronto, 1 King's Circle, Toronto, Ontario, M5S 1A8, Canada.
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  • L. L. Burrows,

    Corresponding author
    1. Department of Biochemistry and Biomedical Sciences, and The Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main St. W., Hamilton, Ontario, L8N 3Z5, Canada
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  • P. L. Howell

    Corresponding author
    1. Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.
    2. Department of Biochemistry, Faculty of Medicine, University of Toronto, 1 King's Circle, Toronto, Ontario, M5S 1A8, Canada.
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E-mail howell@sickkids.ca; Tel. (+1) 416 813 5378; Fax (+1) 416 813 5379;

E-mail burrowl@mcmaster.ca; Tel. (+1) 905 525 9140 x22029; Fax (+1) 905 522 9033.

Summary

Type IVa pili are bacterial nanomachines required for colonization of surfaces, but little is known about the organization of proteins in this system. The Pseudomonas aeruginosa pilMNOPQ operon encodes five key members of the transenvelope complex facilitating pilus function. While PilQ forms the outer membrane secretin pore, the functions of the inner membrane-associated proteins PilM/N/O/P are less well defined. Structural characterization of a stable C-terminal fragment of PilP (PilPΔ71) by NMR revealed a modified β-sandwich fold, similar to that of Neisseria meningitidis PilP, although complementation experiments showed that the two proteins are not interchangeable likely due to divergent surface properties. PilP is an inner membrane putative lipoprotein, but mutagenesis of the putative lipobox had no effect on the localization and function of PilP. A larger fragment, PilPΔ18-6His, co-purified with a PilNΔ44/PilOΔ51 heterodimer as a stable complex that eluted from a size exclusion chromatography column as a single peak with a molecular weight equivalent to two heterotrimers with 1:1:1 stoichiometry. Although PilO forms both homodimers and PilN–PilO heterodimers, PilPΔ18-6His did not interact stably with PilOΔ51 alone. Together these data demonstrate that PilN/PilO/PilP interact directly to form a stable heterotrimeric complex, explaining the dispensability of PilP's lipid anchor for localization and function.

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