A trimeric architecture reveals the glucitol PTS transporter as a distinct superfamily.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates. The glucitol (Gut) PTS transporter from Escherichia coli has often been discussed in relation to the Glucose-Fructose-Lactose (GFL) superfamily, although other work has suggested that it may instead form a separate PTS superfamily. This uncertainty is linked to its unusual genetic organization, in which the transmembrane IIC domain is divided into two polypeptides (IIC1/GutE and IIC2/GutA). Here, we present the cryo-electron microscopy (cryo-EM) structure of the complete Gut transporter, which resolves this discrepancy by revealing a homotrimeric architecture for its transmembrane domain-a fold unprecedented among sugar-transporting PTS permeases. This structural evidence supports the view that the Gut family represents a distinct PTS superfamily. Within the trimer, the protomers are captured in inward-facing and inward-occluded conformations, providing a structural basis for an alternating-access transport mechanism. Furthermore, the structure suggests a unique in-trans phosphotransfer pathway between the IIB and IIC domains of adjacent subunits and identifies the substrate-binding pocket at the GutA/GutE interface. Our work redefines the structural landscape of PTS transporters and provides a mechanistic framework for sugar transport by this unique trimeric porter.