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The pilus usher controls protein interactions via domain masking and is functional as an oligomer.

Werneburg GT, Henderson NS, Portnoy EB, Sarowar S, Hultgren SJ, Li H, Thanassi DG - Nat. Struct. Mol. Biol. (2015)

Bottom Line: Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD).We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher.We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA. [2] Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.

ABSTRACT
The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

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In vivo detection of FimC-FimH binding to the FimDusher(a) Structure of the FimD N domain (blue) bound to a FimC-FimH pilindomain complex (yellow and green, respectively) (PDB ID: 1ZE3, ref. 21). FimD residue Phe4 is depicted in redin stick representation. Phe4 is in close proximity to the FimC chaperone (seealso Supplementary Fig.2a). (b) Structure of the FimD-FimC-FimH complex (PDBID: 3RFZ, ref. 23). FimH is in green,FimC is in yellow, and the FimD domains are colored as in Figure 1. Residues Tyr704 (C1), Thr717 (C1), and Tyr788 (C2)are depicted in red in stick representation. Tyr704 is in close proximity to theFimH adhesin domain, whereas Thr717 and Tyr788 are in proximity to the chaperone(see also Supplementary Fig.2b). (c) SDS-PAGE and immunoblot analysis of invivo, site-directed photocrosslinking. Samples are purifiedHis-tagged WT FimD or FimD Phe4 (N domain), Tyr704 (C1 domain), Thr717 (C1domain), or Tyr788 (C2 domain) amber mutants and associatedcrosslinked products. Additional controls are shown in Supplementary Figure 2.The position of the FimD monomer is indicated on the right for each panel. Theasterisks (*) mark FimD crosslinked products. Uncropped images are shown inSupplementary Data Set2.
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Figure 2: In vivo detection of FimC-FimH binding to the FimDusher(a) Structure of the FimD N domain (blue) bound to a FimC-FimH pilindomain complex (yellow and green, respectively) (PDB ID: 1ZE3, ref. 21). FimD residue Phe4 is depicted in redin stick representation. Phe4 is in close proximity to the FimC chaperone (seealso Supplementary Fig.2a). (b) Structure of the FimD-FimC-FimH complex (PDBID: 3RFZ, ref. 23). FimH is in green,FimC is in yellow, and the FimD domains are colored as in Figure 1. Residues Tyr704 (C1), Thr717 (C1), and Tyr788 (C2)are depicted in red in stick representation. Tyr704 is in close proximity to theFimH adhesin domain, whereas Thr717 and Tyr788 are in proximity to the chaperone(see also Supplementary Fig.2b). (c) SDS-PAGE and immunoblot analysis of invivo, site-directed photocrosslinking. Samples are purifiedHis-tagged WT FimD or FimD Phe4 (N domain), Tyr704 (C1 domain), Thr717 (C1domain), or Tyr788 (C2 domain) amber mutants and associatedcrosslinked products. Additional controls are shown in Supplementary Figure 2.The position of the FimD monomer is indicated on the right for each panel. Theasterisks (*) mark FimD crosslinked products. Uncropped images are shown inSupplementary Data Set2.

Mentions: Ushers are large, integral OM proteins composed of five domains20-23: a periplasmic N-terminal (N) domain, a transmembraneß-barrel channel domain, a plug domain located within the ß-barrelregion, and two periplasmic C-terminal domains (C1 and C2) (Fig. 1 and Supplementary Fig. 1). The N domain provides the initial binding sitefor chaperone-subunit complexes (Figs. 1a and2a)21,24-26. The C1 and C2 domains provide a second bindingsite and anchor the growing pilus fiber (Figs.1a and 2b)23,27,28. In the restingapo-FimD usher, the plug domain occludes the lumen of theß-barrel channel (Supplementary Fig. 1c)20,22,23. The usher must be activated for pilus biogenesisby binding of a FimC-FimH complex to the N domain13,18,29. Activation results indisplacement of the plug to the periplasm, insertion of the FimH adhesin into thechannel lumen, and transfer of FimC-FimH from the usher N domain to the C domains(Fig. 2b)23. The mechanism and specific sequence of eventsdriving usher activation and handoff of chaperone-subunit complexes from the N tothe C domains is not understood. The usher N and C domains bind to the same surfaceof the chaperone, and handoff requires rotation of the chaperone-subunit complex,concomitant with translocation of the pilus fiber through the usher channel towardthe cell surface21,23,26,27. The usher exists in the OM as anoligomer20,28,30,31. However, the pilus fiber issecreted through only one protomer of the usher oligomer, and the usher monomerappears to be sufficient for pilus biogenesis20,23,27,32.Therefore, whether and how the additional usher molecules contribute to thecatalysis of pilus assembly in vivo is a subject of debate.


The pilus usher controls protein interactions via domain masking and is functional as an oligomer.

Werneburg GT, Henderson NS, Portnoy EB, Sarowar S, Hultgren SJ, Li H, Thanassi DG - Nat. Struct. Mol. Biol. (2015)

In vivo detection of FimC-FimH binding to the FimDusher(a) Structure of the FimD N domain (blue) bound to a FimC-FimH pilindomain complex (yellow and green, respectively) (PDB ID: 1ZE3, ref. 21). FimD residue Phe4 is depicted in redin stick representation. Phe4 is in close proximity to the FimC chaperone (seealso Supplementary Fig.2a). (b) Structure of the FimD-FimC-FimH complex (PDBID: 3RFZ, ref. 23). FimH is in green,FimC is in yellow, and the FimD domains are colored as in Figure 1. Residues Tyr704 (C1), Thr717 (C1), and Tyr788 (C2)are depicted in red in stick representation. Tyr704 is in close proximity to theFimH adhesin domain, whereas Thr717 and Tyr788 are in proximity to the chaperone(see also Supplementary Fig.2b). (c) SDS-PAGE and immunoblot analysis of invivo, site-directed photocrosslinking. Samples are purifiedHis-tagged WT FimD or FimD Phe4 (N domain), Tyr704 (C1 domain), Thr717 (C1domain), or Tyr788 (C2 domain) amber mutants and associatedcrosslinked products. Additional controls are shown in Supplementary Figure 2.The position of the FimD monomer is indicated on the right for each panel. Theasterisks (*) mark FimD crosslinked products. Uncropped images are shown inSupplementary Data Set2.
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Figure 2: In vivo detection of FimC-FimH binding to the FimDusher(a) Structure of the FimD N domain (blue) bound to a FimC-FimH pilindomain complex (yellow and green, respectively) (PDB ID: 1ZE3, ref. 21). FimD residue Phe4 is depicted in redin stick representation. Phe4 is in close proximity to the FimC chaperone (seealso Supplementary Fig.2a). (b) Structure of the FimD-FimC-FimH complex (PDBID: 3RFZ, ref. 23). FimH is in green,FimC is in yellow, and the FimD domains are colored as in Figure 1. Residues Tyr704 (C1), Thr717 (C1), and Tyr788 (C2)are depicted in red in stick representation. Tyr704 is in close proximity to theFimH adhesin domain, whereas Thr717 and Tyr788 are in proximity to the chaperone(see also Supplementary Fig.2b). (c) SDS-PAGE and immunoblot analysis of invivo, site-directed photocrosslinking. Samples are purifiedHis-tagged WT FimD or FimD Phe4 (N domain), Tyr704 (C1 domain), Thr717 (C1domain), or Tyr788 (C2 domain) amber mutants and associatedcrosslinked products. Additional controls are shown in Supplementary Figure 2.The position of the FimD monomer is indicated on the right for each panel. Theasterisks (*) mark FimD crosslinked products. Uncropped images are shown inSupplementary Data Set2.
Mentions: Ushers are large, integral OM proteins composed of five domains20-23: a periplasmic N-terminal (N) domain, a transmembraneß-barrel channel domain, a plug domain located within the ß-barrelregion, and two periplasmic C-terminal domains (C1 and C2) (Fig. 1 and Supplementary Fig. 1). The N domain provides the initial binding sitefor chaperone-subunit complexes (Figs. 1a and2a)21,24-26. The C1 and C2 domains provide a second bindingsite and anchor the growing pilus fiber (Figs.1a and 2b)23,27,28. In the restingapo-FimD usher, the plug domain occludes the lumen of theß-barrel channel (Supplementary Fig. 1c)20,22,23. The usher must be activated for pilus biogenesisby binding of a FimC-FimH complex to the N domain13,18,29. Activation results indisplacement of the plug to the periplasm, insertion of the FimH adhesin into thechannel lumen, and transfer of FimC-FimH from the usher N domain to the C domains(Fig. 2b)23. The mechanism and specific sequence of eventsdriving usher activation and handoff of chaperone-subunit complexes from the N tothe C domains is not understood. The usher N and C domains bind to the same surfaceof the chaperone, and handoff requires rotation of the chaperone-subunit complex,concomitant with translocation of the pilus fiber through the usher channel towardthe cell surface21,23,26,27. The usher exists in the OM as anoligomer20,28,30,31. However, the pilus fiber issecreted through only one protomer of the usher oligomer, and the usher monomerappears to be sufficient for pilus biogenesis20,23,27,32.Therefore, whether and how the additional usher molecules contribute to thecatalysis of pilus assembly in vivo is a subject of debate.

Bottom Line: Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD).We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher.We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, USA. [2] Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.

ABSTRACT
The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer-membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate-binding site but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

Show MeSH
Related in: MedlinePlus