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Download Hi-Res ImageDownload to MS-PowerPointCite This:Biochemistry 2013, 52, 17, 2914-2923
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Functional Mapping of PilF and PilQ in the Pseudomonas aeruginosa Type IV Pilus System

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† Program in Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada
‡ Department of Biochemistry, University of Toronto, Toronto, ON, Canada
§ Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada
*P.L.H.: Program in Molecular Structure and Function, The Hospital for Sick Children, 555 University Ave., Toronto, M5G 1X8 ON, Canada; phone, (416) 813-5378; fax, (416) 813-5379; e-mail, [email protected]. L.L.B.: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada; phone, (905) 525-9140, ext. 22029; fax, (905) 522-9033; e-mail, [email protected]
Cite this: Biochemistry 2013, 52, 17, 2914–2923
Publication Date (Web):April 2, 2013
https://doi.org/10.1021/bi3015345
Copyright © 2013 American Chemical Society
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Abstract

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Pseudomonas aeruginosa uses type IV pili (T4P) to interact with the environment and as key virulence factors when acting as an opportunistic pathogen. Assembly of the outer membrane PilQ secretin channel through which the pili are extruded is essential for pilus biogenesis. The P. aeruginosa T4P pilotin, PilF, is required for PilQ outer membrane localization and assembly into secretins and contains six tetratricopeptide (TPR) protein–protein interaction motifs, suggesting that the two proteins interact. In this study, we found that the first four TPR motifs of PilF are sufficient for PilQ outer membrane targeting, oligomerization, and function. Guided by our structure of PilF, site-directed mutagenesis of the protein surface revealed that a hydrophobic groove on the first TPR is required for PilF-mediated PilQ assembly. Deletion of individual domains within PilQ suggests that the N0, KH-like, or secretin domain, but not the C-terminus, interacts with PilF. Purified PilQ was found to pull down PilF from Pseudomonas cell lysates. Together, these data allow us to propose a model for PilF function in the T4P system. PilF interacts directly or indirectly with the PilQ monomer after translocation of both proteins through the inner membrane and acts as a co-chaperone with the Lol system to facilitate transit across the periplasm to the outer membrane. The mechanism of PilQ insertion and assembly, which appears to be independent of the Bam system, remains to be determined.

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Mutants of the PilF hydrophobic groove on TPR1 (A36D, A39D, L43A, L43D, and G46D) compared to wild-type PilF by circular dichroism and the purification of full-length PilQhis and its ability to pull down PilF mutants. This material is available free of charge via the Internet at http://pubs.acs.org.

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