Multiple conformations facilitate PilT function in the type IV pilus

Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C(2) symmetry; however, most of these ATPases crystallize with either C(3) or C(6) symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C(2,) C(3), and C(6) conformations. The physiologic importance of these conformations is validated by coevolution 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/VirB11-like family members.