Abstract
The phosphotransferase system glucose-specific transporter IICB(Glc) serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC(Glc) domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIB(Glc) domain. Furthermore, IIC(Glc) consists of a transport (TD) and a scaffold domain, with the latter being involved in dimer formation. Transport is mediated by an elevator-type mechanism within the IIC(Glc) domain, where the substrate binds to the mobile TD. This domain undergoes a large-scale rigid-body movement relative to the static scaffold domain, translocating glucose across the membrane. Structures of elevator-type transporters are typically captured in either inward- or outward-facing conformations. Intermediate states remain elusive, awaiting structural determination and mechanistic interpretation. Here, we present a single-particle cryo-EM structure of purified, n-dodecyl-β-D-maltopyranoside-solubilized IICB(Glc) from Escherichia coli. While the IIB(Glc) protein domain is flexible remaining unresolved, the dimeric IIC(Glc) transporter is found trapped in a hitherto unobserved intermediate conformational state. Specifically, the TD is located halfway between inward- and outward-facing states. Structural analysis revealed a specific n-dodecyl-β-D-maltopyranoside molecule bound to the glucose binding site. The sliding of the TD is potentially impeded halfway due to the bulky nature of the ligand and a shift of the thin gate, thereby stalling the transporter. In conclusion, this study presents a novel conformational state of IIC(Glc), and provides new structural and mechanistic insights into a potential stalling mechanism, paving the way for the rational design of transport inhibitors targeting this critical bacterial metabolic process.