Multiple conformations facilitate PilT function in the type IV pilus

Abstract

Type IV pilus-like systems are protein complexes that polymerize a fibre of pilins. They are critical for virulence in many pathogens. Pilin polymerization and depolymerization are powered by motor PilT-like ATPases thought to possess C2 symmetry. However, most PilT-like ATPases crystallize with either C3 or C6 symmetry and the relevance of these conformations is unclear. Here we determined the X-ray structures of PilT in four unique conformations and used these structures to classify the conformation of available PilT-like ATPase structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT revealed condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations was validated by co-evolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases. IntroductionType IV pilus-like (T4P-like) systems are distributed across all phyla of prokaryotic life (1, 2). T4P-like systems include the type IVa pilus (T4aP), type II secretion (T2S) system, type IVb pilus (T4bP), Tad/Flp pilus, Com pilus, and archaellum -with the latter found exclusively in Archaea. These systems enable attachment, biofilm formation, phage adsorption, surface or swimming motility, natural competence, and folded protein secretion in Bacteria and Archaea (3-5), and thus are of vital medical and industrial importance. As many of these systems are critical for virulence in bacterial pathogens (1, 6-8), conserved components could have value as therapeutic targets. Despite the importance of T4P-like systems, basic questions -including how the pilus is assembled and disassembled -remain open.All systems have at least three conserved and essential elements: a pilus polymer of subunits termed pilins, a pre-pilin peptidase, and a motor (9). The pre-pilin peptidase cleaves the N-terminal leader peptides of pilin subunits at the inner face of the cytoplasmic membrane, leaving the mature pilins embedded in the membrane (10). Polymerization requires extraction of pilins from the membrane by the cytoplasmic motor using energy generated from ATP hydrolysis (11-13). The motor is made up of two well-conserved components: a cytoplasmic ring-like hexameric PilT-like ATPase and a PilC-like inner-membrane platform protein (9). As mature pilins cannot interact directly with the cytoplasmic PilT-like ATPase, their polymerization requires interaction of both the pilins and ATPase with the PilC-like platform protein (9). Cryoelectron tomography (cryoET) studies of the T4aP, T4bP, archaellum, and T2S systems are consistent with localization of the PilC-like protein in the lumen of the hexameric PilT-like ATPase, connecting it to the pilus on the exterior of the inner membrane (14-17). The PilT-like ATPase connects to stator-like components, suggesting that a fixed PilT-like ATPase moves the PilC-like protein (15). Thus, Pil...

The structure and mechanism of the bacterial type II secretion system

“…Ultimately though, the functional oligomeric state of GspE in vivo has not yet been conclusively shown. Large scale conformational changes were observed between the C6 and C2 symmetry states that were reminiscent of other related ATPases in both the T4PS and archaeal motors (Collins et al, 2018;McCallum et al, 2019;McCallum et al, 2017;Reindl et al, 2013;Satyshur et al, 2007). Overall, the dynamic nature of GspE and its inherent flexibility is suspected to be vital in coupling nucleotide-induced conformational change in the mechanical loading of pseudopilins into the pseudopilus.…”

“…In contrast, how the T4PS ATPase motors extend and retract pili is better understood (Craig et al, 2019) and may provide insight into how the T2SS pseudopilus assembly works given evolutionary relatedness and the likelihood of shared mechanistic principles. In the T4PS a rotary mechanism has been suggested for pilus extension where ATP hydrolysis in the motor PilB is coupled with a clockwise sequence of conformational changes in the hexameric ring (Mancl et al, 2016; McCallum et al, 2019; McCallum et al, 2017; Solanki et al, 2018). These conformational changes may be directly transmitted to PilC (equivalent to GspF in the T2SS) so that it sequentially rotates and shifts 8‒10 Å orthogonally from the membrane toward the periplasm.…”

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

“…Because the putative ATP-binding deficient pilT K136A Walker A mutant exhibited parental levels of piliation ( Fig 1D and 1E ), we hypothesized that the hyperpiliation observed in pilT E204A may be due to the fact that this protein may be locked in an ATP-bound state. A recent preprint shows that different ATP-bound states of PilT exhibit different conformations [31]. This may suggest that the conformation adopted by PilT E204A reduces the ability of PilU to facilitate retraction.…”

Fresh extension ofVibrio choleraecompetence type IV pili predisposes them for motor-independent retraction

“…Thus, motor-independent retraction is likely a conserved feature of diverse T4P. While significant advances have been made in our understanding of motor-dependent retraction (21)(22)(23)(27)(28)(29)(30), a mechanistic understanding of motorindependent retraction and the factors that regulate this process remains lacking.…”