Crystal Structures of the Pilus Retraction Motor PilT Suggest Large Domain Movements and Subunit Cooperation Drive Motility

Abstract

PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.

Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

“…Recent studies of BfpD have shown that while all six members of a BfpD hexamer bind ATP, only alternating subunits interact with the BfpE peptide (Crowther et al, 2005), a finding that is consistent with current models of the pilus as a three-start helix (Ramboarina et al, 2005;Craig et al, 2004Craig et al, , 2006 (Satyshur et al, 2007), showing that heterogeneity in the conformational state of individual subunits within the hexameric assembly is likely to be a common feature of these proteins.…”

“…While the functions of the WA and WB motifs are well characterized, those of the Asp and His Box motifs remain largely unexplored, despite their broad conservation in the secretion NTPase family. Based on limited structural and mutagenesis data, residues of the Asp and His Boxes are likely to contribute to nucleotide binding and/or sensing as well as subunitsubunit interactions (Satyshur et al, 2007;Robien et al, 2003). In this work we demonstrate that the three motor proteins in P. aeruginosa are bona fide ATPases, and we use a site-directed mutagenesis approach to show that for their function all three require specific invariant residues within the characteristic secretion NTPase motifs.…”

“…However, the structure of PilT from A. aeolicus has recently been published (Satyshur et al, 2007), allowing us to use it as a model for the P. aeruginosa motor proteins. The A. aeolicus PilT protein (PilT Aa ), as well as related T2S ATPases EpsE (Vibrio cholerae) and HP0525 (Helicobacter pylori), have a characteristic structure composed of globular N-and C-terminal domains connected by a highly flexible linker domain (Satyshur et al, 2007;Robien et al, 2003;Savvides et al, 2003). The C-terminal domains containing the NTPase motifs are highly conserved among the motor proteins, allowing us to use the ATP-bound form of PilT Aa and the AMP-PNP-bound form of EpsE as structural models with confidence.…”

3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili

“…Whatever aspect of Tfp biology is to be studied, pilus retraction must be taken into account because it generates remarkable mechanical forces (25), likely promoted by domain movements within PilT (26). PilT-powered retraction is indispensable for intimate adhesion (27,28), twitching motility (7,8), competence for DNA transformation (29) and signaling to host cells (30), yet it must be counteracted to achieve efficient biogenesis of pilus filaments.…”

“…E. coli TOP10 (Invitrogen, Carlsbad, CA) was used for cloning experiments, whereas BL21(DE3) was used for protein expression and purification. A methionine auxotroph of the latter strain was used for selenomethionine labeling as described (26,35). To facilitate crystallization, we engineered a strain producing a PilX protein missing the first 28 residues of the mature protein.…”