Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry - PubMed (original) (raw)

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

Rebecca J Rose et al. Proc Natl Acad Sci U S A. 2008.

Abstract

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed "donor-strand exchange (DSE)" in which an N-terminal extension (Nte) of one subunit donates a beta-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

The P pilus, DSC, and DSE complexes and conserved Nte-binding sequences. (A) Schematic diagram of P pilus biogenesis. Subunits (colored), bound to a chaperone (PapD, brown) in the periplasm, assemble at the usher (PapC, gray) in the order PapG, PapF, PapE, PapK, PapA, and PapH. (B) Topology diagram of the DSC interaction. In this complex, the chaperone G1 strand (brown) completes the Ig fold of each subunit (green) by binding into the hydrophobic groove between strands A and F (indicated). The Nte is unstructured. (C) Crystal structure of the DSC interaction between the subunit PapK (shown in space fill) in complex with the chaperone PapD (brown) (PDB ID code 1PDK). The P1–P4 residues and the exposed (unoccupied) P5 pocket are indicated. (D) Sequences of Ntes of different Pap subunits. Full-length Ntes were used for PapA, PapK, PapE, and PapF, whereas the long N terminus of the Nte of PapH [which does not participate in donor-strand exchange (18)] was truncated to aid solubility. The peptides used in this study to mimic each Nte (see Methods) are outlined in black. Residues composing the hydrophobic binding motif (P2–P5) are shown in red. (E) Topology diagram of the DSE interaction. The Nte from one subunit (green) completes the Ig fold of the subunit previously assembled (yellow) by forming a new intermolecular β-strand. (F) Crystal structure of the subunit PapE (shown in space fill) bound to the Nte of PapK (green) (PDB ID code 1N12). The P2–P5 residues that bind to the P2–P5 pockets are indicated.

Fig. 2.

ESI-MS of cognate/noncognate DSE reactions. (A–G) NanoESI mass spectra of the DSE reactions between PapD/PapK and ANte (A–C) and KNte (E–G): 30 min (A and E), 24 h (B and F), and 72 h (C and G) after reaction initiation. The chaperone/subunit complex is shown in red, unbound chaperone in yellow, and subunit/Nte product in blue. (D and H) Relative amount of chaperone/subunit complex (red) and subunit/Nte complex (blue) quantified at different times during DSE by using ESI-MS.

Fig. 3.

Discrimination in pilus assembly revealed by the apparent rate of DSE of different cognate/noncognate chaperone/subunit Nte pairs. (A–F) The apparent rate of DSE, monitored by the loss of chaperone/subunit complex versus time followed by nanoESI-MS for PapD/PapG (A); PapD/PapF (B); PapD/PapENtd1 (C); PapD/PapK (D); PapD/PapANtd1G15N (E), and PapD/PapHNtd1 (F) when challenged with FNte (orange), ENte (yellow), KNte (green), ANte (light blue) or HNte (dark blue). The stability of each chaperone/subunit complex in the absence of Nte peptide is shown in black. Corresponding apparent pseudo first-order rate constants (_k_obs) for each chaperone/subunit Nte pair are shown in (G–K), with cognate interactions indicated (*). For reactions in which <20% of the substrate reacts within 14 days, _k_obs were estimated based on the extent of substrate loss at this time (hatched bars). Errors are given in

Table S1

Fig. 4.

Comparison of the reactivity of each chaperone/subunit complex with its cognate Nte(s). The apparent rate of DSE of each chaperone/subunit complex for all cognate reactions: PapD/PapG plus FNte (red), PapD/PapF plus ENte (orange), PapD/PapENtd1 plus ENte (yellow), PapD/PapENtd1 plus KNte (olive), PapD/PapK plus ANte (green), PapD/PapANtd1G15N plus ANte (light blue) and PapD/PapANtd1G15N plus HNte (dark blue), measured as the loss of the initial chaperone/subunit complex monitored by nanoESI-MS.

Fig. 5.

Chimeric peptides show that the rate of DSE is determined by the C-terminal half of the Nte. (A) Decrease in PapD/PapENtd1 signal intensity when challenged with ENte (yellow), HNte (blue), E/HNte (black), or H/ENte (gray). (Inset) Sequences of ENte, HNte, E/HNte, and H/ENte. (B) Decrease in PapD/PapF signal intensity when challenged with ENte (yellow), KNte (green), E/KNte (black), or K/ENte (gray). (Inset) Sequences of ENte, KNte, E/KNte, and K/ENte. In the sequences, the P2–P5 residues are highlighted in red.

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Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry - PubMed (original) (raw)