Purification and structural characterization of the tularemia membrane protein virulence factors CapB and CapC.

Francisella tularensis, the causative agent of tularemia, relies on the CapBCA membrane protein complex for intracellular survival and virulence, yet none of its components have been structurally characterized. Here, we report successful recombinant expression, membrane targeting, and purification of the CapB and CapC proteins from F. tularensis SCHU S4 using an N-terminal PelB-MBP fusion strategy. Both proteins were efficiently delivered to the inner membrane of Escherichia coli, solubilized in β-DDM detergent, and purified in monodisperse form with yields of approximately 5 mg per 4 L culture. Size exclusion chromatography, Blue Native PAGE, dynamic light scattering, and negative-stain electron microscopy consistently indicated that the maltose-binding protein tagged CapB and CapC each formed detergent-stabilized oligomers. Circular dichroism spectroscopy showed predominantly α-helical secondary structure content for both proteins and revealed differential sensitivity to ionic strength. Structural predictions using AlphaFold 3 and membrane-interaction modeling support the presence of one transmembrane helix for CapB and five for CapC, consistent with their hydropathy profiles. These results provide the first biochemical and structural characterization of CapB and CapC, enabling future high-resolution studies of the CapBCA complex and advancing efforts to understand its role in F. tularensis virulence and potential therapeutic targeting.