Ins and outs of major facilitator superfamily antiporters

Abstract

The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters.

Figure 1

3D structures of GlpT, EmrD and OxlT viewed parallel to the membrane. (a) The 3.3 Å structure of GlpT in the C_i conformation with the transmembrane α-helices of the N-terminal domain colored magenta, and those of the C-terminal domain in green. The substrate-translocation pore is situated between the two domains (35). (b) The GlpT substrate-binding site, depicting the basic residues intimately involved in binding (Lys 80, Arg 45, His 165 and Arg 269) and those that participate in intra- and interhelical salt bridge formation (Lys 46, Asp 274 and Glu 299). (c) The 3.5 Å structure of EmrD in a compact, occluded state (83). (d) The 6.5 Å density map of OxlT, also in an occluded state, with 12 TMs modeled into it (30). In all of the above, the periplasmic side of each transporter is at the top.

Figure 2

Substrate-binding and transport cycle of GlpT. (a) Schematic diagram illustrating the salt bridges that are formed and broken upon initial loose and subsequent tight binding of substrate to GlpT. TM1, 7 and 8 are depicted as cylinders, and the amino acid side chains that participate in salt bridge formation are depicted as stick models. The substrate molecule is represented as a filled circle. When substrate binds loosely to GlpT in the C_i conformation, and H165 is unprotonated, interhelical salt bridges are formed between R45-D274, and K46-D274 (44). (b) Protonation of H165 elicits tighter substrate binding, and R45 and R269 move closer to each other, pulling TM1 and TM7 closer together. The R45-D274 interhelical salt bridge breaks and a new intrahelical one forms between R269 and E299. The existing interhelical salt bridge formed between K46 and D274 becomes stronger, and the transporter takes on a more compact conformation (44). (c) Schematic diagram of the single binding site, alternating access mechanism with a rocker-switch type of movement for the GlpT-mediated G3P-P_i exchange reaction. The diagram describes the proposed conformational changes that the transporter undergoes during the reaction cycle. C_o represents the protein in the outward facing conformation and C_i the inward facing one. The G3P substrate is represented by a small disk and triangle, and P_i is represented by a small disk (35). (d) A schematic free energy diagram illustrating the energy levels of the different conformations of GlpT that occur during the transport reaction cycle under physiological conditions. In the absence of substrate-binding the energy barrier prevents the conformational interconversion between the C_o and C_i states of the transporter. Substrate binding lowers the energy barrier sufficiently to allow Brownian motion (kT) to drive the conformational interconversion. The energy barrier is represented by a dotted line. S denotes substrate (43).