The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters

Abstract

Crystal structures of the bacterial amino acid transporter LeuT have provided the basis for understanding the conformational changes associated with substrate translocation by a multitude of transport proteins with the same fold. Biochemical and modeling studies led to a "rocking bundle" mechanism for LeuT that was validated by subsequent transporter structures. These advances suggest how coupled solute transport might be defined by the internal symmetry of proteins containing inverted structural repeats.

FIGURE 1. Alternating-access mechanisms

A simple mechanism for alternately exposing a substrate binding site to either side of the membrane involves interconversion through an occluded intermediate (top left). This mechanism is sufficient to explain transport of a single solute (uniport), two or more solutes in the same direction (symport), and movement of solutes in opposite directions (antiport). For efficient symport, the interconversion of outward- and inward-facing transporter conformations cannot occur when only one substrate is bound, and for efficient antiport the interconversion can occur only when one of the substrates is bound.

FIGURE 2. Cytoplasmic pathway of SERT and repeat structure of LeuT

A: Cytoplasmic pathway of serotonin transporter SERT is lined by amino acid residues (spheres) from transmembrane helices 1 (red), 5 (pale green), 6 (green), and 8 (cyan). The model of SERT was built by homology to LeuT and is viewed from the plane of the membrane with the backbone trace in ribbons, with bound serotonin (yellow), chloride (magenta), and sodium ions (dark blue) shown as spheres. B–D: the LeuT fold contains inverted topology repeats of five trans-membrane helices. Repeat A contains transmembrane helices 1–5 (B), and repeat B consists of transmembrane helices 6–10 (C), according to the LeuT numbering. Superposition (D) of the last three transmembrane helices from each of those repeats (pale colors) indicates a relative difference in the orientation of the first two transmembrane helices (dark colors). These two conformations of the repeats may contain the essence of the two alternating-access states of the transporter.

FIGURE 3. Comparison of LeuT X-ray structure and cytoplasm-facing model

A: a model of the cytoplasm-facing conformation of leucine transporter, LeuT (right), constructed by swapping the conformations of the two five-transmembrane-helix repeats, compared with the X-ray crystal structure bound to leucine (left), viewed from the plane of the membrane, showing how the bundle (red or yellow) pivots around the central binding site at approximately the position of Ser256. B: residues found to be accessible to cysteine-modifying reagents in serotonin transporter (red or orange) are not exposed in the extracellular-facing X-ray structure of LeuT but become exposed in the cytoplasm-facing model. The primary motion is an outward rotation of the cytoplasmic half of the bundle (light green), with respect to the scaffold (light blue); for reference, two residues (Arg-5 and Asp-367) are colored dark green and orange, respectively. Structures are viewed from the cytoplasmic side of the membrane. Leucine (yellow) and sodium (blue) bound at the central binding site are visible at the base of the cytoplasmic pathway in the model but not in the X-ray structure.

FIGURE 4. Comparison of LeuT with vSGLT

The cytoplasmic pathway in LeuT-fold structures, viewed from the cytoplasm. A model of LeuT in a cytoplasm-facing conformation (center), generated by swapping the conformations of the two five-transmembrane repeats in LeuT (left), differs from the X-ray structure of LeuT (left) but resembles that of a structure of vSGLT (right), also in a cytoplasm-facing conformation. Structures are shown with helices as cylinders, showing how the cytoplasmic halves of TMs 1 (red), 2 (orange), 6 (green), and 7 (blue) in the bundle peel away from the scaffold (gray) in the cytoplasm-facing conformations but are packed against the scaffold in the extracellular-facing conformation of LeuT (left).

FIGURE 5. Rocking of the bundle within the LeuT transporter fold structure results in opening and closing of pathways on either side of the membrane

The surface of the scaffold region from LeuT is shown as a blue cut-away surface, viewed from the plane of the membrane. The axis of the four-helix bundle consisting of TMs 1, 2, 6, and 7 is shown as a line, colored from red for extracellular-facing through green for cytoplasm-facing conformations. Axes were defined as the line joining the average coordinates of all helical residues in the upper and lower leaflets of the membrane, after superposition of the residues in the scaffold onto those of LeuT. Four-helix bundles are shown as cartoons for the X-ray structures of tryptophan-bound LeuT (dark red) and for vSGLT (dark green), which are in the fully open extracellular-facing and cytoplasm-facing conformations, respectively.