CHARACTERIZATION OF THE TYPE IV PILUS MOTOR COMPLEX

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Type IV pili are long thin filaments displayed on the surfaces of many bacteria. They have a diverse array of functions that rely on their ability to (i) adhere to various substrates, including host cell surfaces, pili from nearby bacteria, DNA and bacteriophage, and (ii) to depolymerize or retract, which pulls the bacteria along mucosal surfaces, pulls them close together in protective aggregates, and can draw substrates like DNA and phage into the bacterium for nutrition and genetic variation. Type IV pili are polymers of thousands of copies of the major pilin subunit. The pili are assembled at the inner membrane by a complex molecular machine spanning the bacterial envelope from the cytoplasm to the outer membrane. Our lab is at the forefront of this field, with our x-ray crystal structures of Type IV pilin subunits and cryo-electron microscopy reconstructions of intact pilus filaments providing an atomic resolution picture of these organelles and a framework for understanding filament assembly. Remarkable progress has been made by many groups in understanding the architecture of the pilus assembly apparatus, yet the mechanism by which these pili are assembled remains a mystery. This proposal aims to characterize the molecular motor complex within the pilus machinery to reveal how it drives filament assembly. Type IV pili are assembled from pilin subunits, which are anchored in the inner membrane via their hydrophobic N-terminal alpha-helices prior to pilus assembly. Polymerization is powered by a hexameric ring-shaped assembly ATPase on the cytoplasmic side of the inner membrane. ATP hydrolysis induces a conformational change in individual protomers of the ATPase, which is relayed to the growing pilus through a polytopic inner membrane platform protein, resulting in incremental extrusion of the filament from the membrane upon each subunit addition. These critical components together form the motor complex. We hypothesize that sequential ATP hydrolysis in the ATPase protomers alters their affinity for the individual platform protein domains, causing them to “walk” around the ATPase ring. This circular motion of the platform protein around the base of the growing pilus brings its small periplasmic loop into contact with the bottom of the pilus, extruding it outward a short distance as it passes. An incoming pilin subunit immediately fills the newly opened gap at the base of the pilus, preventing the pilus from collapsing back into the membrane. We will test this rotary pilus assembly model by characterizing the components of the motor complex by x-ray crystallography, examining their interactions both in vivo and in vitro, and testing for rotation of the motor components using optical tweezers. We will focus on the Vibrio cholerae toxin-coregulated pilus machinery as it is one of the simplest and best characterized of the Type IV pilus systems.