Structure of ASBT

Structure Overview:

A ribbon reperesentation of ASBT

X-ray crystallography has determined that the transporter is made up of cytoplasmic N and C termini, along with 10 transmembrane helices that are connected by short loops.

The domains:

The transmembrane helices are assembled into two domains, and the conformation of the transporter depends on their positioning:

·         The panel domain

·         The core domain

The panel domain is made up of transmembrane helices 1, 2, 6, and 7. The core domain is made up of the remaining helices. Helices 4 and 9 are discontinuous, forming hairpins, and cross over each other where they are broken (they will be referred to as helices 4a and 4b, and 9a and 9b).

ASBT and NhaA are the only Na²⁺/H⁺ transporters that  have discontinuous helices crossing over each other. The structural homology was even greater when the panel and core domains were examined separately. Although they transport different substrates of varying sizes (NhaA is a sodium and proton antiporter) they still had a structural plan that was common for both transporters, meaning that the structure must be highly flexible. ^[1]

Sodium binding sites:

ASBT is a sodium symporter, meaning that it will move Na²⁺ ions across the membrane in the same direction as the bile acid.

By studying the structure it has been found that there are two Na²⁺ binding sites; one between the TM4b and TM5 helices, near to the cytoplasmic surface, and is known to interact with the Glu260 residue found in the TM9a helix. The other binding site is located where TM4 and TM9 cross.
Side chains that surround the two binding sites are highly conserved in ASBT and NTCP (ASBT’s liver equivalent). The Glu260 residue previously mentioned has been found to be important in the function of them both. When it was replaced with an alanine residue bile acid transport was reduced.

Two Na²⁺ binding site is needed as the second Na²⁺ will neutralise a negative dipole that is present in the TM9a helix, this increases the helix’s stability. Using this information we can assume that two sodium ions are needed per bile acid molecule. ^[2][3]

.
The red spheres are sodium ions, showing where the two
 sodium binding sites can be found, in the core domain

Binding the substrate:

Substrate enters via the cavity circled in red

 The side of the transporter that is facing into the lumen of the ileum has a gap between the two domains allowing the Na²⁺ ions and bile acid molecules to diffuse in. This gap is created by a bend in the N-terminal region of TM1 helix. The other side of the transporter is closed by TM1, 2, 4b, 7, 9b and 10.

Asn295 shown in TM10

Many different substances can be recognised by ASBT due to its unusually large cavity.
This cavity is mostly hydrophobic (bile acids are impermeable ions at physiological pH) , but at the end of the cavity exist polar residues and water molecules. TM10 has a large role in substrate transport as it forms a weak hydrogen bond between the substrate and its Asn295 residue. By mutating Asn295 residue to alanine, translocation decreases by 80%.

The two conformations of ASBT:

ASBT has two conformations; inwards and outwards.^[4] But how does the transporter change from one conformation to another? By looking at NhaA we see that Na²⁺ binding causes the conformational change, and as the two proteins are structurally similar, ASBT probably works the same way.

outward and inward conformations^[5]

Both Na²⁺ binding sites are in the core domain with the second binding site positioned where the two discontinuous helices cross.