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2JLN is one of the conformations of Mhp1, which is a membrane secondary transporter.^[1]

Function

Mhp1 is a trans-membrane protein bellowing to the nucleobase-cation-symport-1 (NCS1) transporter family from Microbacterium liquefaciens.

A secondary transporter, like Mhp1, effects the cellular uptake and release of a wide range of substances across biological membranes in all organisms. This is done by coupling the uphill movement of the substrate against its concentration gradient with the energetically favorable downhill gradient of a second substrate, often a proton or a cation. The kinetics and thermodynamics of the transporters can be explained by their alternating conformations.Cite error: Invalid <ref> tag;

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In the case of Mhp1, it allows the sodium dependent income of indolyl methyl- and benzyl-hydantoins (Figure 1) in the cell. Those are part of a salvage metabolic pathway leading to their conversion in amino acids.

2JLN is one of the conformations of Mhp1. It is the outward-facing conformation without substrate.

Figure 1 : Structure of benzyl-hydantoin, the substrate of Mhp1

Relevance

Hydantoins are important compounds in salvage pathways for nitrogen balance in yeasts and plants and are particularly interesting commercially for the synthesis of chiral amino acids.

Transporters from the NCS1 family are also important in the toxicity of the antifungal agent, 5‐flucytosine and mutations in the proteins can lead to drug resistance. Mhp1 is an excellent model system for elucidating how substrates or inhibitors, including drugs, are recognised at the molecular level and then taken up into cells by members of the NCS1 transporter family.

Mhp1 is of more general significance because it is also structurally homologous to other proteins in different subfamilies of the superfamily of secondary transporters. The study of the mechanisms of Mhp1 enables the understanding of other members of this family too. Members of the neurotransmitter‐sodium‐symport family (NSS), solute‐sodium‐symporter family (SSS) and amino acid‐polyamine‐organocation family (APC) are secondary transporters similar to Mhp1. They play important roles in human physiology, being responsible for the accumulation of molecules such as neurotransmitters, sugars, amino acids and drugs into cells.^[2]

Disease

Dysfunction of members of the transporters family in humans can lead to diseases including neurological and kidney disorders. Other members are implicated in cancer as they can supply tumor cells with nutrients, cause drug resistance and/or provide a means of treatment.Cite error: Invalid <ref> tag; name cannot be a simple integer. Use a descriptive title

Structural highlights

Overall structure

The protein is composed of one chain of 12 transmembrane alpha helices (TMs). They are organised in two repeating units connected by a 59-residue loop (TMs 1-5 and TMs 6-10) and two additional helices (TM 11 and 12). The two repeating units have a symmetrical topology (Figure 2).

Figure 2 : Structure of Mhp1 from Microbacterum liquefaciens

The central bundle is composed of TMs 1 and 2, twined to the TMs 6 and 7 respectively. In addition, the protein presents a V-shape structure formed by TMs 3 to 5, twined to TMs 8 to 10 (Figure 2).

TM5 and TM10 are 'flexible helices' because they bend during the state transitions.^[3]

The substrate- and cation-binding sites are located in the space between the central four-helix-bundle and the outer helix layer.

Structure of the substrate binding site

A large cavity on the inward facing side of the protein is made up of the neighboring surfaces of TMs 1, 3, 5, 6 and 8. This cavity connects the substrate and cation binding sites.

The substrate binding site is located at the break of the TMs 1 and 6. (Figure 2)

The substrate, the benzyl-hydantoin interacts with the amino acids of the binding site. The hydantoin group establisches pi-stacking interactions with the indole ring of Trp 117 on TM3 and Trp 220 on TM6 and hydrogen bonds with Asn 318 and Gln 121. The benzyl ring interacts with Trp 220 and Gln 42 (Figure 3).

Figure 3 : Substrate binding site

Structure of the cation binding site

Mhp1 is a sodium dependent protein. The sodium binds at the C-terminal end of TM1a and interacts with TM8 (Figure 2). The dipole moment at the C-terminus of TM1a contributes to the binding.

Experiments have shown that sodium increases the affinity of benzyl-hydantoin for Mhp1 and reciprocally benzyl-hydantoin increases the affinity of sodium for Mhp1.

Indeed, the presence of benzyl-hydantoin in Mhp1 binding site blocks the pathway of the sodium ion to the extracellular side.^[4]

Therefore, the binding of the substrate and the cation are closely coupled.

Conformational states

Mhp1 exists in two conformational states depending if the substrate is bound or not: the substrate free structure (-BH), corresponding to the outward-facing open and the substrate bound structure (+BH), corresponding to the outward-facing occluded (Figure 4).

Figure 4: The conformational change upon the substrate binding

The binding of the substrate induces conformational changes of the protein allowing it uptake in the cell.

Figure 5 : Proposed substrate translocation mechanism by Mhp1

The binding of the substrate in the binding site leads to a switch from the outward-facing open state (2JLN) to the outward-facing occluded state (4d1b). The TM10 arrangement changes (Figure 4.C) and closes the access to the “OUT” side space of the membrane (Figure 5.A).

Then, there is a change from the outward-facing occluded state to the inward-facing occluded state (4d1a). (Figure 5.B) The substrate-binding site is occluded from the inside of the membrane. It seems that the movement involves the helix bundle of TMs 3 and 8. Moreover, researchers are working on the possibility of a coordinated shifting of TMs 1 and 6 shift with TMs 3 and 8.

Eventually, there is a switch from the inward-facing occluded state to the inward-facing open state (2x79). This allows the release of the substrate in the cytoplasm. However, the structures involved in the change still be unclear (Figure 5.C).