The production of extracellular proteinaceous filaments, called pili, is a nearly ubiquitous adaptation used by bacteria and archaea to sense and interact with their environment. In Caulobacter crescentus, the tight adherence Tad pili (type IVc) undergo dynamic cycles of extension and retraction, but Tad pili lack a retraction ATPase ortholog. Mutations in the CpaF ATPase result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction. I will present our recent progress towards an improved understanding of the molecular details of Tad pili structure-function using single particle cryoEM and AlphaFold3 molecular modeling. These approaches revealed conformational changes in the ATPase CpaF that explain how bidirectional activity is achieved, which was validated by mutations that disfavor the retraction conformation. Furthermore, in situ structure determination of the Tad pilus, co-immunoprecipitation, and molecular models point to an activity switching mechanism. Tad pili are part of a large family of evolutionarily related filament systems, including the type II secretion system, gram-positive competence pilus, archaeal pili, and the archaellum, of which many employ only a single motor ATPase. Thus, these results may serve as a blueprint to decipher the biosynthetic mechanisms of other single motor filament production systems across bacteria and archaea.

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