EJEMPLOS DEL SIGLO XXI O EL VALOR DE USO SOMETIDO

The publication of the first high-resolution structure of the SLC6 family ancestor LeuT caused a revolution in understanding the structure and substrate binding of B0AT1 and B0AT3 [192]. Subsequent crystal structures of LeuT [193-196], MhsT from B.halodurans [197], the D. melanogaster dopamine (DAT) [198], and human serotonin (SERT) [199] SLC6 transporters in various confirmations, has allowed for a full transport cycle to be proposed [200]. A homology model based on LeuT showed B0AT1 was composed of 12 TM α-helices [201]. Sequence homology and structural conservation shows B0

AT1 and B0AT3 contain the pseudo two-fold inverted axis of symmetry common to all known SLC6 transporters (Fig 1.5). The axis of symmetry demonstrates that helices 1-5 and 6-10 represent a repeated fold, which can be superimposed by inverting and overlaying them (Fig. 1.6). The two-fold inverted-repeat fold is a structural architecture shared by several other transporter families [202-205]. The remaining two helices, TM11 and 12, are less conserved across the SLC6 family but retained in all human members, including B0AT1 and B0AT3 [201].

Based on this structural conservation, B0AT1 and B0AT3 consist of two subdomains (Fig. 1.7, central panel). The first two helices of each repeat motif form the ‘bundle’ subdomain (helices 1/6 and 2/7). A second ‘hash’ subdomain formed by the 3rd and 4th helices in each inverted repeat (helices 3/8 and 4/9) constitutes a relatively rigid scaffold around the bundle. During the outward to inward transition of the transport cycle, the bundle is hypothesised to bend at unwound regions in the middle of TM1 and 6 [194]. The unwinding is caused by 3 consecutive conserved glycine residues and makes available the functional groups of several nearby residues for substrate binding as they are not required for α-helix hydrogen bonding [206]. This results in a ‘rocking’ or hinge-bending movement of TM1 and 6 outwards into the bilayer between a gap formed between TM helices 5 and 7 [95, 194, 207]. The proposed

29 SLC6 transporter mechanism will be elaborated on in the context of B0AT1 and B0AT3 interactions with ancillary proteins in the discussion (section 6.2.3).

Bundle symmetry is also reflected in the conservation of SLC6 substrate and ion binding sites in B0AT1 and B0AT3. Amino acid carboxyl- and amino-group, first sodium binding (Na1), and chloride binding sites are formed by residues in bundle helix pairs 1/6 and 2/7 (Fig. 1.7A and 1.7B). Amino acid side-chain and second sodium site (Na2) binding residues are located predominately in the hash domain helices TMs 3/8 and 1/8, respectively (Fig. 1.7C). All substrate carboxylate and amine co-ordinating atoms (≤ 3.5 Å) are conserved in LeuT, Drosophila DAT, human SERT and B0-like transporters. Where residue identity is not conserved, the actual co-ordinating atom is unchanged. For example, A22 and T254 in LeuT are C49 and S278 in B0AT1, co-ordinating substrate in all cases by the main chain carbonyl (Cα=O).

A pattern emerges in B0AT1 and B0AT3 binding sites, where differences in substrate-ion stoichiometry and substrate specificity cannot be easily explained because of the high level of residue conservation. For example, B0AT1 has conserved Na1, Na2, and substrate binding sites like other SLC6 transporters, but a transport stoichiometry of 1 Na+:1 substrate. In contrast, B0AT3’s conserved Na1, Na2, substrate, and chloride binding sites are consistent with its 2 Na+: 1Cl−: 1 substrate stoichiometry. Likewise, as substrate specificity from B0AT1 to B0AT3 changes, involving shortening of the amino acid side chain length, no differences are observed in the side-chain binding pocket residues. Two substrate side-chain stabilising residues in mouse B0AT3, N420 and V110, differ in functional mouse B0AT3 and non- functional human B0AT3 (T421 and I111) – possibly contributing to the non-functionality of the human transporter.

30

31 Figure 1.5 Sequence alignment and conservation in SLC6 Broad Neutral Amino Acid Transporters

Sequence alignment of human B0AT1 (UniProt ID Q695T7), human B0AT3 (Q96N87), mouse B0AT1 (Q9D687), mouse B0AT3 (O88576), and SLC6 homologues, the Drosophila melanogaster dopamine transporter DAT (Q7K4Y6) and LeuT from Aquifex aeolicus (O67854). The alignment was created and verified using PROMALs3D [208], with structural alignment filtering using the x-ray crystal structures of LeuT (PDB 2A65) and DAT (4M48) [209]. Substrate and ion binding residues are coloured as indicated in the figure legend. TM helices are numbered and sit above the MSA corresponding to the amino acids which form them. Note: although conservation of substrate and multiple ion binding sites is shown, this does not necessarily reflect the observed transport stoichiometry of the individual transporters, only the potential conservation of such binding sites.

33 Figure 1.6 Membrane topology of B0AT1 and B0AT3 and repeat motif symmetry Top: membrane topology and orientation of B0AT1 and B0AT3. The two repeated 5 x 5 halves of the transporter are separated by a dashed line. Symmetric helices are paired by colour: 1and 6 (blue), 2 and 7 (grey), 3 and 8 (green), 4 and 9 (pink), 5 and 10 (maroon). Helices 11 and 12 do not form part of the pseudo two-fold inverted repeat motif, but are present in both B0AT1 and B0AT3. Helices 1 and 6 are separated into two halves by short unwound regions in the middle of the membrane. Bottom: The 2nd repeat motif (faded) is inverted, rotated 180° about the axis of symmetry (top) and overlaid on the first repeat motif to demonstrate the pseudo two-fold axis of asymmetry. The α-helices of all SLC6 transporters with elucidated structures have the same conserved repeat motif [191, 193].

35 Figure 1.7 B0AT1 and B0AT3 conserved substrate and ion binding sites

Central Figure: The homology model of human B0AT1 is based on the outward open (competitive inhibitor bound) conformation of the Drosophila melanogaster dopamine transporter DAT (PDB ID 4m48). The bundle (cyan) and hash (magenta) sub-domains, along with TM5 (yellow) and part of IL4 (blue) are coloured. TM helices are numbered 1-12. Sodium ions (purple) and chloride (red) are represented as ionic radii spheres. L-leucine (green) is represented as a stick structure. Close up panels: Binding sites are constructed from homology models of human B0AT1, mouse B0AT3, and x-ray crystal structures for DAT or LeuT (PDB ID 2A65). Binding residues involved in A) chloride binding, B) Na1 plus L-leucine amine and carboxylate binding, and C) Na2 plus L-leucine side chain binding. The three panels for each binding site show details from the B0AT1, B0AT3 and DAT or LeuT outward occluded binding sites. Binding residues are coloured to reflect their bundle (cyan) or hash (magenta) origin and labelled with residue identity, number and intermolecular bond length (Å). All oxygen (red) and nitrogen (blue) atoms involved in intermolecular hydrogen bonding or dipole-ionic interactions are represented by dashed lines (- - - - -), in C) all labelled residues without a dashed line are involved in L-leucine side chain Van der Waal interactions. Hydrogen atoms have been removed for clarity of viewing. ? denotes the possibility the Na2 site in B0AT1 is occupied by a sodium ion.

36 The translocation of only one sodium ion in B0AT1 is mysterious given the high conservation in LeuT, DAT, B0AT1, and B0AT3 sodium binding sites. Even where conservation is not absolute, for example B0AT1 residues C49 (Na1) and S431 (Na2), neither C49 nor S430 change the sodium co-ordinating atom. Interestingly, when SLC6 transporters are chloride- independent, such as B0AT1, only one sodium ion is translocated [100] (see Fig. 1.4). In addition, a close functional association between chloride binding and the Na2 site has been observed for the dopamine transporter DAT (SLC6A3) [210].

B0AT1-mediates chloride-independent transport [39, 40], with F74 representing the only change in the conserved SLC6 chloride binding site. A polar side-chain equivalent to B0AT1C49, generally replacing an alanine, is also exclusive to chloride-independent SLC6 members, NTT4, B0AT2, and B0AT1, and PROT (SLC6A7), whose ion stoichiometry is not known [100]. These differences perhaps represent an alternative binding mode and transport mechanism to chloride-dependent SLC6 transporters. Brӧer [167] has hypothesised the pH- sensitive B0AT1 [39, 171] has a hydroxyl binding site in place of chloride, to aid isomerisation from inward facing to extracellular facing during the transport cycle. Recent structural and molecular dynamics evidence suggests protonation of Glu290 in LeuT is a key event for inward to outward facing transition in the space that later evolved into a chloride binding site in SLC6 members [200, 211]. Forming part of the B0AT1 Na1 binding site, the cysteine sulfhydryl proton from C49 could act to stabilise a hydroxyl anion. In contrast B0AT3, which is chloride-dependent, has the conserved alanine (A34) in this position, and is pH-insensitive [106]. What exact role, however, chloride plays in the general SLC6 transport mechanism is still unclear [212-215].

37 An ordered substrate-first [40] and random binding order [39] were proposed for B0AT1 based on the Michaelis-Menten assumption that the maximal transport rate (𝑉_𝑚𝑎𝑥) for any single steady-state substrate concentration can be modulated by changing the concentration of the substrate that binds last [190]. However, the appearance of a random binding order from kinetic transport data could also represent the interdependence of amino acid and sodium binding, as has been shown for other SLC6 homologues where sodium at Na1 forms a co- ordination ligand for the substrate (see Fig. 1.7B, [192, 194, 216]). Evidence from SLC6 and other secondary active transporters indicates that binding of the driving substrate(s), always ions, proceeds uphill substrate binding [195, 214, 215, 217-219]. Furthermore, no outward open SLC6 transporter crystal structures have been found bound with substrate alone, while several have been solved binding only sodium [193, 194, 198]. Based on this evidence it is likely B0AT1 and B0AT3 exhibit ordered binding, with sodium ions binding prior to, or as a co-ordinated pair with, amino acids.