Evidence for the coupling of substrate recognition with transporter opening in MOP-family flippases

Abstract

Bacteria produce a variety of surface-exposed polysaccharides important for cell integrity, biofilm formation, and evasion of the host immune response. Synthesis of these polymers often involves the assembly of monomer oligosaccharide units on the lipid carrier undecaprenyl-phosphate at the inner face of the cytoplasmic membrane. For many polymers, including cell wall peptidoglycan, the lipid-linked precursors must be transported across the membrane by flippases to facilitate polymerization at the membrane surface. Flippase activity for this class of polysaccharides is most often attributed to MOP (Multidrug/Oligosaccharidyllipid/Polysaccharide) family proteins. Little is known about how this ubiquitous class of transporters identifies and translocates its cognate precursor over the many different types of lipid-linked oligosaccharides produced by a given bacterial cell. To investigate the specificity determinants of MOP proteins, we selected for variants of the WzxC flippase involved in Escherichia coli capsule (colanic acid) synthesis that can substitute for the essential MurJ MOP-family protein and promote transport of cell wall peptidoglycan precursors. Variants with substitutions predicted to destabilize the inward-open conformation of WzxC lost substrate specificity and supported both capsule and peptidoglycan synthesis. Our results thus suggest that specific substrate recognition by a MOP transporter normally destabilizes the inward-open state, promoting transition to the outward-open conformation and concomitant substrate translocation. Furthermore, the ability of WzxC variants to suppress MurJ inactivation provides strong support for the designation of MurJ as the flippase for peptidoglycan precursors, the identity of which has been controversial.\n\nSIGNIFICANCEFrom cell walls in bacteria to protein glycosylation in eukaryotes, surface exposed polysaccharides are built on polyprenol-phosphate lipid carriers. Monomer units are typically assembled at the cytoplasmic face of the membrane and require translocation to the cell surface for polymerization/assembly. MOP-family proteins are a major class of transporters associated with this flippase activity. Despite their ubiquity and importance for cell surface biology, little is known about their transport mechanism. Here, we investigated substrate recognition by MOP transporters in bacteria. We present evidence that transport proceeds via destabilization of the inward-open state of the transporter by specific substrate binding thereby promoting a transition to the outward-open state and substrate release on the opposite face of the membrane.