Abstract
Extracellular glycan biosynthesis is a widespread microbial protection mechanism. In Gram-negative bacteria, the O antigen polysaccharide represents the variable region of outer membrane lipopolysaccharides. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the WzmWzt ABC transporter for ligation to the lipopolysaccharide core sugars. In agreement with its role as a polysaccharide transporter, WzmWzt forms a continuous transmembrane channel in a nucleotide-free state.
Here, I present the high-resolution structures of two distinct conformations of ATP-bound Aquifex aeolicus WzmWzt. In Chapter 2, I provide the crystal structure of ATP-bound WzmWzt and compare this conformation with the crystal structure of nucleotide-free WzmWzt. Large structural changes within the nucleotide-binding and transmembrane regions push conserved hydrophobic residues at the substrate entry site towards the periplasm and provide a model for polysaccharide translocation. With ATP bound within the nucleotide binding domains, the transporter forms a large transmembrane channel, with openings toward the membrane and periplasm, that is wide enough to accommodate an O antigen polysaccharide. The channel’s periplasmic exit is sealed by detergent molecules that block solvent permeation. In a molecular dynamics (MD) simulation of water flux, an O antigen-occupied transporter is sealed, preserving the membrane potential. Further MD analyses suggest that, in a biological membrane, lipid molecules occupy the channel’s periplasmic exit and prevent water flux in the transporter’s resting state, leading to the proposal of a lipid-gating hypothesis.
In many cases, O antigen transport by WzmWzt depends on the chemical modification of the O antigen’s non-reducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). In Chapter 3, I present the cryo-electron microscopy structure of the full-length WzmWzt transporter bound to ATP and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD protomer. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a novel closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters. Together, these analyses further our understanding of the WzmWzt transport mechanism culminating in a revised model of processive transport for the channel-forming O antigen transporter WzmWzt.
