High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na+ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of similar to 0.5 nm, similar to 1.0 nm, and similar to 1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh. Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.

Automated summary

This study used high-speed atomic force microscopy to visualize the transport cycle of the CitS protein in real time. It revealed the presence of three distinguishable states and independent movement of the protein subunits. These findings provide insights into the transport mechanism of the CitS protein.