Inward-Open

For the LeuT transporter to open up on the inside, it undergoes a series of conformational changes. The main transmembrain domains (TMs) involved are TM1, TM2, TM5, TM6, TM7, and the extracellular loops (ELs) are moved as a consequence.


Some of the domains tilt and some bend to cause the opening and closing.

The following picture shows the conformation of the inward-open transporter.

Figure 1-Structure of inward-open conformation of LeuT

From superimposing the inward-open structure on the outer-occluded structure, it is found that:

• TM1a rotates by about 45° and goes from pointing downwards to the cytosol to pointing into the membrane.
• TM6b rotates by 17° away from the central binding site
• TM1b and TM6a rotate by 24° and 21° respectively towards the scaffold domain. This blocks the extracellular pathway.
• TM2, TM7 and TM5, which support TM1 and TM6, bend rather than tilt as whole rigid structures, and the bending is facilitated by a Gly or Pro residue in the middle. This is shown by the fact that their intracellular parts and extracellular parts rotate by different degrees.
• The rotation of the extracellular part of TM7 by 17° results in the dipping down of EL4 into the extracellular vestibule. This closes off the extracellular solvent pathway.
• TM11 moes away from the centre so TM6a can roate towards the scaffold domain.

Gating

A thick extracellular gate is formed in the inward-open state to prevent access from solvents on the extracellular side, and the thick intracellular gate opens to expose substrate binding site on the inside.

If you are interested, here are the some interactions between amino acids that are involved!

Extracellular gate

These interactions formed lead to the closing of the extracellular gate in the inward-open conformation of LeuT:

· Between Arg30 on TM1b and Asp404, Gly408, Thr409 on TM10

· Arg30 (TM1b) which is on top of Phe253 (TM6a) makes several interactions with TM10 including direct salt bridge with Asp404, hydrogen bonds to Thr 409 and carbonyl oxygen of Gly408.

· Between Ala319 on EL4 and Asp401 on TM10

· Between Asp240 on TM6a and Tyr471 on TM11

Intracellular gate

Interactions in the thick intracellular gate of outward-facing and outward-occluded structures are disrupted rather than formed to open the intracellular gate, and these are the interactions disrupted:

·         Ionic interaction the N-terminus and TM1a (Arg5, Trp8) forms with TM6b (Ser267, Tyr 268) and TM8 (Gln361, Asp369) on the intracellular side.

·         Between Lys 196 and Thr10.

·         Between Ser 278 and Arg11.

·         The last two disruptions are caused by the spreading of TM5, TM7 and TM1a.

These findings on how the interactions in the extracellular gate and intracellular gate affect the gating mechanism are quite promising, as consistent results are found by mutating homologous amino acid residues in GAT-1 (sodium and chloride-dependent GABA transporter in humans) and some eukaryotic transporters respectively.

Collapsing

In the inward-open conformation, the extracellular vestibule of LeuT collapses. This helps to block extracellular pathway. The collapsed extracellular vestibule leads to the opening of the intracellular gate.

The S2 substrate binding site also collapses in this inward-open state, which is suggested by the deeply buried residues Ile111 and Leu400 lining the S2 site.

Hinges

As mentioned earlier, some helices bend rather than tilt as a rigid structure, and these domains have hinges to help with the bending, just as doors have hinges. The bending of the domains allows the opening of the inside and the closing up of the outside of the transporter.

Here are some details.

· The hinge for TM1a is at Leu25. This is above the Na2 site, separating the residues on TM1a and TM8 defining the Na2 site. 

· This means that the release of Na⁺ from that site, the movement of TM1a and the opening up to the inside are coupled. 

· Without sodium ion, the intracellular pathway opens when TM1a rotates away from the scaffold domain and the sodium ion sites are disrupted.

· It is also suggested that the tilting of TM5 is needed for TM1a to be in the rotated position, and the tilting of TM5 pushes TM7 and TM1b, which closes the extracellular gate.

· There are 2 hinges for TM6, Ser256 for TM6a and Phe259 for TM6b. They are close to the substrate binding site but far from Na1 site, which means weakening the Na1 site but little disruption at the substrate binding site

· TM3 and TM8 which interact with the substrate remains in the same position despite several effects caused by changes in TM1 and TM6.

· These tell us that the substrate binding pocket is maintained in this inward-open conformation and the transporter can still bind substrate.