Structural and functional investigations of type IV pilus biogenesis proteins

Abstract

Type IV pili are retractable fibres found on the surface of a wide variety of Gram-negative bacteria. They mediate diverse functions, including host-cell adherence, surface motility, biofilm formation and DNA uptake. More than a dozen different proteins are implicated in type IV pilus biogenesis; many have homologues in the type II secretion system which suggests that the two systems are evolutionary linked. Although type IV pilus biogenesis and retraction have been predominantly studied at the genetic level in Gram-negative bacteria, little is understood about the way in which ATP hydrolysis in the cytoplasm is coupled to the assembly and disassembly of the pilus fibre in the periplasm, and also to the uptake of DNA. The PilT retraction ATPases from Neisseria meningitidis, Vibrio cholerae and Thermus thermophilus were purified and characterised; however, their poor solubility precluded structural studies. Research was then focused on the integral inner membrane protein PilC from Thermus thermophilus. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis provided evidence that PilC forms a tetramer. The structure of full length PilC was determined using cryo electron microscopy at 20 A resolution. The structure has a bilobed asymmetric shape with a "fin-like" top and "cone-like" bottom, connected by a narrow "waist". Limited proteolysis analysis demonstrated that the N-terminus of PilC is unstable and susceptible to proteolytic cleavage. Therefore, a truncated version of PilC, with this part of the N-terminus removed, was expressed, purified and used in biochemical studies. The assembly of PilC was studied using site-directed mutagenesis analysis and size-exclusion chromatography coupled with multi-angle laser light scattering. This provided evidence that the dimer interface of the crystal structure of the PilC N-terminal domain reported previously, also occurs in the intact PilC protein and, therefore, the PilC tetramer adopts C2 symmetry. Topology analysis using a green fluorescent protein fusion and nanogold labelling suggested that the N- and C- domains of PilC are located in the cytoplasm and periplasm respectively. Collectively, these results provide evidence that PilC is a transmembrane protein which is exposed on both sides of the inner membrane, providing potential binding surfaces for interaction with other type IV pilus machinery components. The ATPase PilF is required for high transformation efficiency in Thermus thermophilus, although the mechanism by which it mediates this function is unknown. Here, PilF from Thermus thermophilus was shown to bind both single and double stranded DNA, as well as RNA, with high affinity. The structure of PilF was determined by cryo electron microscopy at 18 A resolution. It comprises two stacked ring/disc-like regions with C6 symmetry, which were assigned to the C- and N- terminal domains respectively. A model is been suggested for DNA uptake by PilF, in which ATP hydrolysis causes structural changes in the C-domains which are transferred to the N-domains to take up DNA into the cell. This is the first report of 3D structure of a type IV pilus ATPase associated with transformation and DNA uptake. These results have contributed to a structural understanding of some of the components of the type IV pilus biogenesis system, paving the way for better understanding at the molecular level in the future.

Date of Award	1 Aug 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeremy Derrick (Supervisor)