Biochemical, structural and functional analysis of bacterial polysaccharide co-polymerase proteins
Polysaccharide co-polymerase (PCP) proteins are involved in the biosynthesis of several bacterial surface polysaccharides, including lipopolysaccharide O antigens and capsular polysaccharides (CPSs). PCP family members are integral inner membrane proteins that are characterized by a proline motif and a conserved membrane topology. In addition, secondary structure analyses predict highly conserved structural elements. Despite theses similarities, PCP proteins are classified into three sub-families based on their involvement in different polysaccharide assembly and export pathways. PCP-1 (Wzz) homologs determine O antigen chain length in Wzy-dependent biosynthesis pathways. PCP-2a homologs form an envelope-spanning complex with a partner outer membrane polysaccharide export (OPX) protein and are required for polymer assembly and export in Wzy-dependent CPS biosynthesis pathways. PCP-3 proteins are involved in ABC transporter-dependent CPS biosynthesis pathways and are also thought to form an export channel with a partner OPX protein. However, the functional relationships between the three sub-families are unclear. The data presented in this thesis show the structural characterization of PCP-1 and PCP-3 homologs embedded in lipid bilayers using EM and image analysis techniques. These experiments revealed that PCP-1 homologs share a conserved hexameric organization. A 14 A resolution projection map was generated for a representative PCP-1 homolog (WzzST) from images of two-dimensional crystals obtained by cryo-EM. The PCP-1 projection map was consistent with the structure of a PCP-1 hexamer modeled from the available crystal structure data. Cryo-EM of a PCP-3 homolog (CtrB) showed that this subfamily also forms conical structures that extend from the lipid bilayer and are consistent with the structures of PCP-1 and PCP-2a proteins. Site-directed fluorescence labeling of CtrB showed that it possesses two transmembrane spans that separate a central periplasmic region, in contrast to previous reports in the literature for PCP-3 proteins. Functional complementation studies were used to show that CtrB operates specifically with its OPX partner protein (CtrA). These data support a model for PCP-3 proteins in which they form the inner membrane proximal component of an export complex. This would be comparable to the model proposed for PCP-2a and their partner OPX proteins. Together, these studies extend the understanding of structural and functional relationships between PCP proteins. They are expected to be critical in the design and implementation of future experiments that test models for bacterial polysaccharide assembly and export.