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

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 ␤-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.donor strand exchange ͉ electrospray ionization ͉ noncovalent protein complexes ͉ uropathogenic bacteria

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

“…The resolving power of the technique is often sufficient to monitor posttranslational modifications (such as glycosylation and phosphorylation) and small ligand binding on intact protein complexes up to the megadalton range (18,58,61). Data acquisition takes on the order of minutes between samples, allowing kinetic measurements of relatively slow reactions in the minutes-to-hours timescale (65). Studies on amyloid formation and chaperone and virus assembly illustrate how multiple co-occurring states can be separated, detected, and characterized simultaneously with native MS (66)(67)(68).…”

The Structure of the PapD-PapGII Pilin Complex Reveals an Open and Flexible P5 Pocket

“…The intact PapD-GII chaperone-adhesin complex serves as the initiator of P-pilus synthesis by binding to the usher, PapC (33,66). Further, the PapGII pilin domain's relatively low rate of DSE allows priming of pilus assembly, which proceeds to completion once PapG and PapF have undergone DSE (51). This study completes the structural inventory of P-pilus pilin domains and provides further insight into the mechanisms by which DSE occurs.…”

“…Likewise, there is a high affinity of the tip-associated adhesin of the type 1 pilus to its usher (41,56). In light of the two PapD-GIIp crystal structures and the results of the molecular dynamics studies presented here, the slow DSE of PapGIIp in vitro (51) suggests that catalysis of DSE by PapC is likely to accelerate exchange to the rate seen in vivo, where pili assemble in minutes (26). Indeed, while a completely closed P5 pocket in the case of the terminator PapH (64) is likely to prevent DSE, a P5 pocket that fluctuates between the open and closed states on a nanosecond time scale should allow protein-protein interactions if subunits are found in close proximity and in a permissive orientation, e.g., at the usher.…”

“…Repeating monomers of PapA arranged in a right-handed helical cylinder form the homopolymeric pilus rod (3,14,63). PapA and PapE Ntes are able to complete the fold of other PapA and PapE Ig-like domains, respectively, allowing formation of linear homopolymers (14,51). In the case of PapA, surfaces on pilin then engage in further interactions that promote the coiling of the linear PapA fiber into a rigid helical rod consisting of ϳ3.3 PapA subunits per turn (14).…”

Molecular mechanism of bacterial type 1 and P pili assembly

“…However, pilus subunit ordering is not solely determined by the affinities of the chaperonesubunit complexes for the usher's NTD but depends also on the DSE rate between subunits. In vitro studies have been carried out on the DSE rates between cognate and non-cognate subunit pairs in both the Fim and the Pap systems and both with and without usher [55][56][57]. By 'cognate', we mean here pairs of adjacent subunits interacting with each other as observed in the mature pilus.…”