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 C 2 symmetry. However, most PilT-like ATPases crystallize with either C 3 or C 6 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 C 2, C 3 , and C 6 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 C 2 conformation. With these data we propose a comprehensive model of PilT function with broad implications for PilT-like ATPases.