Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex |
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Authors: | L.M. Sampaleanu M. Ayers S. Tammam S.K. Burley L.L. Burrows P.L. Howell |
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Affiliation: | 1 Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 2 New York SGX Research Center for Structural Genomics (NYSGXRC) & Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA 3 Department of Biochemistry and Biomedical Sciences and The Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 4 Department of Biochemistry, Faculty of Medicine, 1 King's College Circle, University of Toronto, Toronto, Ontario, Canada M5S 1A8 5 Eli Lilly and Company (formerly SGX), 10505 Roselle Street, San Diego, CA 92121, USA |
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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. |
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Keywords: | T4P, type IV pili T2S, type II secretion PDB, Protein Data Bank CC, coiled coil BSA, buried surface area |
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