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

Abstract

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.

Structural basis of CHMP2A–CHMP3 ESCRT-III polymer assembly and membrane cleavage

“…The membrane coated tubes were immobilized at the surface using streptavidin on top of a lipid bilayer (DOPC) on mica containing 0.01% biotinylated lipid 77 . HS-AFM images were analyzed using Igor Pro, and ImageJ with additional home written plugins 78 . Height measurements were performed on raw images after tilt correction.…”

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

“…In support of this idea, a recent structure of CitS, another homodimeric elevator transporter in a different structural family, reveals a CitS dimer with one inward-facing and one outward-facing protomer (Kim et al, 2017), and a recent high-speed atomic force microscopy study of the CitS elevator translocation motion reported that CitS undergoes non-cooperative interprotomer conformational dynamics (Maity et al, 2022). Combined with evidence that Glt Ph , an elevator-type, glutamate transporter that unlike VcINDY is from a thermophilic organism, also shows independence between the motions of its three protomers (Akyuz et al, 2013; Erkens et al, 2013; Ruan et al, 2017), these results suggest that such independence may be a general feature of oligomeric elevator transporters (Garaeva and Slotboom, 2020).…”

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

“…HS-AFM experiments were done using RIBM (Japan) machine in amplitude modulation tapping mode in liquid 23,24,67,68 . Short cantilevers USC-F1.2-k0.15 (NanoWorld, Switzerland) with a spring constant of 0.15 N/m, resonance frequency around 0.6 MHz, and a quality factor of ~2, and ultra-small cantilevers BL-AC10FS-A2 (Olympus, Japan) with a spring constant of 0.1 N/m, and resonance frequency around 0.6 MHz in buffer were used.…”