# Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation

## TL;DR

This study reveals a new intermediate structure of a bacterial glucose transporter, offering insights into its mechanism and potential drug targets.

## Contribution

The first cryo-EM structure of IICBGlc in an intermediate conformation is presented, revealing a stalling mechanism.

## Key findings

- The IICGlc transporter is trapped in a previously unseen intermediate state using a specific ligand.
- The ligand binding and thin gate shift are identified as factors that stall the transporter's movement.
- This structure provides a foundation for designing inhibitors targeting bacterial glucose transport.

## Abstract

•Glucose transport in bacteria is facilitated by the IICBGlc transporter.•The cryo-EM structure of IICGlc in an intermediate state is reported.•A ligand-induced wedging stalls the elevator-type transport cycle.•This paves the way to the development of specific inhibitors for IICBGlc.

Glucose transport in bacteria is facilitated by the IICBGlc transporter.

The cryo-EM structure of IICGlc in an intermediate state is reported.

A ligand-induced wedging stalls the elevator-type transport cycle.

This paves the way to the development of specific inhibitors for IICBGlc.

The phosphotransferase system glucose-specific transporter IICBGlc serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IICGlc domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIBGlc domain. Furthermore, IICGlc 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 IICGlc 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 IICBGlc from Escherichia coli. While the IIBGlc protein domain is flexible remaining unresolved, the dimeric IICGlc 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 IICGlc, 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.