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

Sourav Maity, Gianluca Trinco, Pedro Buzón, Zaid R Anshari, Noriyuki Kodera, Kien Xuan Ngo, Toshio Ando, Dirk J Slotboom*, Wouter H Roos

*Bijbehorende auteur voor dit werk

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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 ∼0.5 nm, ∼1.0 nm, and ∼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.

Originele taal-2English
Artikelnummere2113927119
Aantal pagina's9
TijdschriftProceedings of the National Academy of Sciences of the United States of America
Volume119
Nummer van het tijdschrift6
DOI's
StatusPublished - 8-feb-2022

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