Structure and elevator mechanism of the Na + -citrate transporter CitS

Abstract

The recently determined crystal structure of the bacterial Na-citrate symporter CitS provides unexpected structural and mechanistic insights. The protein has a fold that has not been seen in other proteins, but the oligomeric state, domain organization and proposed transport mechanism strongly resemble those of the sodium-dicarboxylate symporter vcINDY, and the putative exporters YdaH and MtrF, thus hinting at convergence in structure and function. CitS and the related proteins are predicted to translocate their substrates by an elevator-like mechanism, in which a compact transport domain slides up and down through the membrane while the dimerization domain is stably anchored. Here we review the large body of available biochemical data on CitS in the light of the new crystal structure. We show that the biochemical data are fully consistent with the proposed elevator mechanism, but also demonstrate that the current structural data cannot explain how strict coupling of citrate and Na transport is achieved. We propose a testable model for the coupling mechanism.

Structural basis for the reaction cycle of DASS dicarboxylate transporters

“…Such a mechanism ensures tightly coupled import of Na + and substrate in cotransporters, while the reversible binding of Na + allows for the concentration of the divalent substrate against its electrochemical gradient. Similar charge compensations mechanism have been proposed for the citrate transporter CitS and the glutamate transporter EAAC1 42,43 . coli whole cells following a published protocol with minor modifications 19,45 .…”

Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS

“…In essence we propose that CitS proteins employ a sodium dependent elevator-like movement in which the dimerization domain is held fixed during the transport cycle, and the transport domain moves up and down across the membrane bilayer to translocate the substrate. Further research is required to unravel many details of this model, such as how substrate transport by 2-HCT proteins is coupled to the ion gradient and/or ion binding 52 .…”

ECF-Type ATP-Binding Cassette Transporters

“…Mutations allowed for destabilization of the interface, which made this protein amenable to study the homodimerization of a polytopic membrane protein (110,111). However, because ClC-ec1 has a single interface, this system does not allow for the study of dynamic reorientation of membrane protein interfaces, which frequently occurs in transport proteins-for example, in the glutamate transporter homolog Glt Tk , the citrate transporter CitS, and the succinate transporter vcINDY-in which a transport domain slides along a static scaffold domain (80)(81)(82)(83)112). Glt Ph has been used to study the kinetics of intraprotein movement by a single-molecule fluorescence approach, but the molecular determinants that allow these dynamic transitions are not understood (113).…”

“…In transporters using the elevator mechanism, the transport domain (equivalent to the S-component) moves through the membrane to carry the substrate from one side to the other, and a scaffold domain (equivalent to ECF-T) provides a stable scaffold along which the movement can take place. In the glutamate transporter family, the transport domains occlude the substrate completely (as in S-components of group II), whereas in other cases (such as in CitS or vcINDY) the substrates are not completely occluded by the transport domain and face the scaffold domain during transport (as may be the case in YkoE) (45,(79)(80)(81)(82)(83). A difference between the elevator mechanism and the toppling mechanism is that toppling involves mostly rotation of the S-component, with a minor translational component, whereas the converse takes place in elevator-like movements (79,80,84,85).…”