Alternating access mechanisms of LeuT-fold transporters: trailblazing towards the promised energy landscapes

Abstract

Secondary active transporters couple the uphill translocation of substrates to electrochemical ion gradients. Transporter conformational motion, generically referred to as alternating access, enables a central ligand binding site to change its orientation relative to the membrane. Here we review themes of alternating access and the transduction of ion gradient energy to power this process in the LeuT-fold class of transporters where crystallographic, computational and spectroscopic approaches have converged to yield detailed models of transport cycles. Specifically, we compare findings for the Na⁺-coupled amino acid transporter LeuT and the Na⁺-coupled hydantoin transporter Mhp1. Although these studies have illuminated multiple aspects of transporter structures and dynamics, a number of questions remain unresolved that so far hinder understanding transport mechanisms in an energy landscape perspective.

Figure 1. Inverted repeat and motifs of ligand recognition and binding in representative LeuT-fold transporters

Ligand-bound crystal structures of LeuT (PDB 2A65), Mhp1 (PDB 4D1B), BetP (PDB 4AIN chain b) and CaiT (PDB 2WSX chain a) are shown with helices colored similarly to illustrate conservation of overall topology as well as substrate (cyan) and ion (magenta) binding sites. Helices and Na⁺ are designated numerically using LeuT nomenclature. Ion binding sites are substituted by residue side chains in CaiT. Superimposition of helices composing the inverted repeat (IR, bottom left of each panel) can be achieved by an approximate 180° rotation of one repeat (IR2) in the membrane plane.

Figure 2. Alternating access models derived from crystal structures

(a) Inward-facing (IF, left) and outward-facing (OF, right) conformations of LeuT (PDB 3TT3 and 3TT1), Mhp1 (PDB 2X79 and 2JLN), and BetP (PDB 4LLH chain c and b) are shown. Scaffold helices 3, 4, 8, and 9 are shown as a grey surface within the outline of the complete protein structure. Bundle helices 2 and 7 and EL4 are shown as colored surface to demonstrate degree of bundle rotation. Bundle helices 1 and 6 are depicted as colored cartoons. The extracellular and intracellular vestibules are shown in cyan. (b) The core motif of the bundle for outward-facing and inward-facing conformations is shown after alignment along TM helices 2 and 7 to display deviations from rigid body movements. RMSD calculations are shown for each alignment.

Figure 3. Spectroscopic analysis of Mhp1 alternating access supports the Rocking Bundle model

Outward-facing (OF, PDB 2JLN) and inward-facing (IF, PDB 2X79) crystal structures are shown with relevant helices depicted in cartoon representation. Distances measured by DEER between sites of interest are indicated by a line connecting black spheres that identify spin label locations. The corresponding distance distributions are shown in two middle panels for TM7 (extracellular) and TM1 (intracellular) under apo (black), Na⁺-bound (blue), and Na⁺/substrate-bound (red) conditions, emphasizing shifts in conformational equilibria between closed and open states (grey). As seen in the four right panels, these EPR-derived states are correlated to distributions calculated from each of the three available crystal structure conformations: outward-facing occluded (OFO, PDB 2JLO), outward-facing (OF), and inward-facing (IF). Predicted distance distributions from crystal structures were calculated by the Molecular Dynamics of Dummy Spin Labels (MDDS) approach [50,51].

Figure 4. Ligand-dependent changes in distance distributions deviate from the crystallographic model of alternating access

Outward-facing (OF, PDB 3TT1) and inward-facing (IF, PDB 3TT3) crystal structures are shown with relevant helices depicted in cartoon representation. Distances measured by DEER between sites of interest are indicated by a line connecting black spheres that identify spin label locations. Colors correspond to those in Figure 3. Distance distributions shown in the top panels for TM6 on either side of the membrane underscore ligand-dependent conformational equilibria. The bottom six panels compare the EPR-derived closed/open states to distributions calculated from each of the three crystal structure conformations of LeuT: outward-facing occluded (OFO, PDB 2A65), outward-facing (OF), and inward-facing (IF). Predicted distance distributions from crystal structures were calculated by the Multiscale Modeling of Macromolecular systems (MMM) approach [52].