Structural cycle of the Thermus thermophilus PilF ATPase: the powering of type IVa pilus assembly

Richard Collins,Angela Thistlethwaite,C Alistair Siebert,Jeremy P Derrick,Rana Dajani,Vijaykumar Karuppiah

doi:10.1038/s41598-018-32218-3

Richard Collins, Angela Thistlethwaite + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-32218-3

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Abstract

Type IV pili are responsible for a diverse range of functions, including twitching motility and cell adhesion. Assembly of the pilus fiber is driven by a cytoplasmic ATPase: it interacts with an inner membrane complex of biogenesis proteins which, in turn, bind to nascent pilin subunits and mediate fiber assembly. Here we report the structural characterization of the PilF TFP assembly ATPase from Thermus thermophilus. The crystal structure of a recombinant C-terminal fragment of PilF revealed bound, unhydrolysed ATP, although the full length complex was enzymatically active. 3D reconstructions were carried out by single particle cryoelectron microscopy for full length apoprotein PilF and in complex with AMPPNP. The structure forms an hourglass-like shape, with the ATPase domains in one half and the N1 domains in the second half which, we propose, interact with the other pilus biogenesis components. Molecular models for both forms were generated: binding of AMPPNP causes an upward shift of the N1 domains towards the ATPase domains of ~8 Å. We advocate a model in which ATP hydrolysis is linked to displacement of the N1 domains which is associated with lifting pilin subunits out of the inner membrane, and provide the activation energy needed to form the pilus fiber.

Highlights

Type IV pili (TFP) are widespread in Gram negative bacteria, including several notable human pathogens[1,2]
TtPilFc is identical in sequence but in a different crystal form to a structure published previously for a similar TtPilF fragment derived from a different Thermus thermophilus strain[18]
Inspection of all 6 binding sites revealed electron density consistent with bound nucleotide; there was no evidence for ATP hydrolysis in any site (Supplementary Fig. S2)

Summary

Introduction

Type IV pili (TFP) are widespread in Gram negative bacteria, including several notable human pathogens[1,2]. The TFP assembly ATPases are part of the extensive AAA+ superfamily which form molecular motors, using the energy from ATP hydrolysis to drive mechanical transformations[14]. Within this superfamily, PilF shares a number of sequence similarities, in terms of its domain organisation, with GspE, an ATPase which provides the energy for type II secretion. Including the outer membrane secretin and the PilMNO inner membrane platform[1,2] These parallels are suggestive of a similar fundamental mechanism operating in the two secretion systems. The results are consistent with studies carried out on related ATPases which indicate that the relative orientations between the two domains vary widely[19]

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