Substrate free Outward-open

In the substrate-free sodium-bound state, the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. More specifically, a consequence of hinge-like movements in TMs 1b, 2a, and 6a, relative to the outward-occluded state. Sodium ions bridge interactions between the intracellular halves of the core and scafoold domains, thus keep the occlusion of intracellular thick gate or assume the outward-open conformation. [11]. When LeuT^k is substrate-free, it adopts an outward-open conformation. The substrate forges interaction between the core and scaffold domains, this constrains TM1b and TM6a to move inward, thus assuming an outward-occluded conformation. TMs 1b, 2a and 6a pivot at Val 23, Gly 55 and Leu 257, respectively, suggesting that when substrate no longer forges interactions between the core and scaffold domains, constraints on TM1b and TM6a are released, allowing them to move outward and the transporter to adopt an outward-open conformation.

Extracted from X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Krishnamurthy H, Gouaux E. Nature. 2012 Jan 9;481(7382):469-74. doi: 10.1038/nature10737

Figure 1 shows the sodium-bound outward-open and the Na-leucine-bound outward-occluded conformations. Outward-open structure is coloured with Na1 ions as purple spheres. The outward-occluded structure with Na1 ions (spheres) is grey and leucine is shown in stick representation.  Rupture of extracellular gate interactions (grey dashed lines) in the outward-open structure. Two water molecules that bridge Arg 30 and Asp 404 in the outwardoccluded state are shown as red spheres.

In outward-occluded state, Arg 30 forms a water-mediated salt bridge with Asp 404 (TM10), forming a ‘thin’ extracellular gate [12]. When the water-mediated salt bridge ruptures which separates TM1b/TM6a and TM3/TM10, a pathway to the extracellular environment is opened. [12] The outward-open structure suggests that the presence of sodium ions keeps the intracellular thick gate closed by bridging interactions between the intracellular halves of the core and scaffold domains [12].

Extracted from X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Krishnamurthy H, Gouaux E. Nature. 2012 Jan 9;481(7382):469-74. doi: 10.1038/nature10737

Binding of Na1 at this site may precede substrate binding in order to stabilise an intracellular-closed conformation. Figure 2a and 2b show sodium sites in the outward-open states. The Na1 site involves stabilizing local conformations of TMs 1 and 6 and engaging the main-chain carbonyl oxygen of

Ala 22 (TM1a) with side-chain oxygens of Asn 27 (TM1b) and Thr 254 (TM6a). The Na2 site is located towards the TM1 intracellular region, engaging TM1a and TM8 helices with their main-chain carbonyl of Gly 20 and Val23 of TM1a. Thus, the binding of sodium ions stabilise the inward-occluded conformation.