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

Volkan, Ender, Ford, Bradley A., Pinkner, Jerome S., Dodson, Karen W., Henderson, Nadine S., Thanassi, David G., Waksman, Gabriel, Hultgren, Scott J.
Proceedings of the National Academy of Sciences of the United States of America 2012 v.109 no.24 pp. 9563-9568
Escherichia coli, Gram-negative bacteria, fimbriae, mechanism of action, tissues
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.