Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling

Abstract

SummaryTypes 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod’s remarkable mechanical properties and illuminates its role in pilus secretion.

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

“…Overall, P pili are ~81 Å in diameter with a hollow lumen of ~21 Å diameter. This atomic model of the P pilus explains the mechanism of rod uncoiling at the molecular level by revealing the details of an extensive inter-subunit interaction network 50 . As previously shown, the DSE interactions connecting adjacent subunits are strong hydrophobic interactions, which are topologically essential as they provide the fold-complementing interactions for each subunit in the polymer 20,21,51 .…”

“…Recently, structures were determined for the type 1 pilus by solid state NMR 49 and for the P pilus to ~3.8 Å resolution by cryo-electron microscopy (cryo-EM) 50 , showing a similar overall organisation. The structure of the P pilus in its coiled state shows a right-handed superhelix of 3.28 PapA subunits per turn, an axial rise of 7.7…”

“…As previously shown, the DSE interactions connecting adjacent subunits are strong hydrophobic interactions, which are topologically essential as they provide the fold-complementing interactions for each subunit in the polymer 20,21,51 . By contrast, the remainder of the subunit interaction network responsible for maintaining the helically wound quaternary structure of the pilus is not only composed of predominantly weak hydrophilic contacts but is also topologically non-essential as it is not involved in subunit folding and stability 50 . These distinct sets of interactions explain how CU pili can gradually uncoil by first breaking the weaker hydrophilic interactions between the subunit stacks that form the pilus quaternary structure when an external force is applied, but retain the integrity of the polymer by virtue of the strong hydrophobic and topologically essential DSE interaction.…”

Cryo-EM of bacterial pili and archaeal flagellar filaments

“…While negative stain and cryo-EM [21] have been used in the past to study these pili, the resolution has been too low to allow for any detailed understanding of the interactions that provide the unusual mechanical properties of these filaments: in the presence of forces, these pili can unwind into a thin strand that is five times the length of the normal coiled filament [22]. Using a direct electron detector, cryo-EM images were obtained which allowed for a reconstruction of the P pilus at 3.8 Å [23] with the legacy SPIDER package [14] and the Iterative Helical Real Space Reconstruction algorithm [24]. The donor strand from one subunit is shown inserted into a β-sheet of a neighboring subunit in Fig.…”

Electron Bio-Imaging Centre (eBIC): the UK national research facility for biological electron microscopy

“…The data collected at eBIC have, at the time of writing, generated ten research publications (Hospenthal et al, 2016;Serna et al, 2016;Joseph et al, 2016;Wilkinson et al, 2016;Iadanza et al, 2016;Ramsay et al, 2016;Fica et al, 2017;Swuec et al, 2017;Ilangovan et al, 2017;Boland et al, 2017). We have also received a number of personal communications from inhouse and external users reporting reconstructions at better than 4 Å resolution, with a few extending beyond 3 Å .…”