Coxiella burnetii is an obligate intracellular bacterial pathogen that causes Q fever in humans and animals. Unlike other intracellular bacterial pathogens, C. burnetii occupies a unique autolysosomal niche termed the Coxiella containing vacuole (CCV). The establishment of the CCV is necessary for C. burnetii survival and replication within host cells. C. burnetii utilises an arsenal of over 130 type IV effector proteins (T4Es) some of which modulate host cell trafficking pathways allowing for the formation of the CCV. Cig57 (Cbu1751), Cbu1752, and Cbu1754 are three novel Coxiella T4Es implicated in CCV biogenesis through the subversion of host clathrin-mediated transport. Cig57 interacts with the host protein FCHO2, a nucleator of clathrin-mediated transport. A Cig57/FCHO2 interaction re-routes clathrin to the CCV with unknown effect, and is thought to be mediated by endocytic sorting motifs present on Cig57. Cbu1752 and Cbu1754 are hypothesised to act synergistically with Cig57, based on the ability of Cig57, Cbu1752, and Cbu1754 to co-immunoprecipitate. Although Cig57, Cbu1752, and Cbu1754 have been identified as T4Es implicated in CCV expansion, their mechanistic function and relationship with each other and the clathrin mediated transport system as a whole remains unknown.
Using a combination of analytical ultracentrifugation (AUC) and small angle x-ray scattering (SAXS) experiments, the basic structural characteristics of Cig57, Cbu1752, and Cbu1754 were determined. Cig57 and Cbu1754 are monomeric and roughly globular in solution, whereas Cbu1752 adopted an extended conformation and displayed low levels of self-association. The crystal structure of the Cig57 central domain (residues 178-307) was solved with a resolution of 1.75 Å. The central domain of Cig57 adopts a 4-helix bundle and reveals two hypothetical endocytic sorting motifs form the hydrophobic core of Cig57, indicating that these previously described 'motifs' are highly unlikely to mediate interactions and instead serve to stabilize the Cig57 structure. This work is the first to structurally characterise any Coxiella protein, leading to a better understanding of novel effector protein function and clathrin mediated transport.