LeuBAT mutants

Further study of BATs would  allow further development of new drugs to treat depression, but it is difficult to obtain a 3-dimensional structure of these molecules using X-Ray crystallography. Using the bacterial homologue of BATs, The difference in affinity of BATs and LeuT was overcome by genetically engineering the LeuT protein by mutating key residues found in the primary binding pocket which are involved in substrate binding. It is known that tryptophan binds to LeuT at the primary binding pocket as a competitive inhibitor. The key residues in the primary binding pocket of the LeuT/tryptophan (Figure 1a) complex and LeuT/hSERT (human serotonin transporter) complex (Figure 1b) were used in an amino acid sequence alignment. From this alignment it was possible to determine 20 residues which point towards the primary binding pocket and are divergent from SERT. These residues had previously been determined to be important in SERT pharmacology. This information was used to create the LeuBAT mutant. The “first shell” residues and the “second shell” residues were mutated and the binding constant (Kd) of the two therapeutic agents paroxetine and mazindol were subsequently measured. A list of LeuBAT mutants and their respective mutated residues can be found in table 1. LeuBAT mutants Δ5 and Δ6 have similar Kd values for paroxetine and mazindol. The final version of LeuBAT, Δ13, had a Kd value of 431 nM for paroxetine (SSRI) and 112 nM for mazindol. The second value is very similar to that of SERT, which is 103 nM. Sertraline had the highest affinity for Δ13 with a Kd value of 18 nM.  Comparing these Kd values with the ones obtained with the LeuT wild-type shows how important these key residues are for substrate binding in the primary binding pocket. The LeuT wild-type had a Kd of 308 nM for sertraline and 22.3 nM for mazindol, while paroxetine had a very low affinity that could not be measured. The Δ13 mutant has the most mutated residues among the LeuBAT mutants and is structurally very similar to SERT. 

Thus, Δ13 was used to find out the structural basis of interaction between inhibitors and BATs. Since the binding position of inhibitors is similar between theΔ6 andΔ13 construct (figure 2), higher resolution structures of the Δ6 complexes were obtained for analysis to validate the drug-binding sites in Δ13.

There are still some differences between LeuBAT and SERT. One of them is the position of one of the sodium ions, Na1, which binds to different residues in each complex. In the wild-type Na1 is bound to the α-carboxyl group of Leu and Trp, while in the  Δ13 mutant this position is occupied by a water molecule which allows the formation of a salt bridge between Na1 and Asp24. This stabilization of the structure with a sodium ion contributes to a higher affinity for the therapeutic agents.

A

B

Figure 1. (a)The representation of mutation positions around the primary binding pocket in the wild-type LeuT–Trp structure. Bound tryptophan (green stick）and the mutated residues are in sticks. The transmembrane helices TM1, TM3, TM6, TM8 and TM10 around the pocket are highlighted as red, green, blue, yellow and magenta, respectively. The extracellular region and intracellular region are distinguished by two with sticks. (b) Sequence alignment of wild-type LeuT and human serotonin transporter (hSERT). It shows the regions of TM1, TM3, TM6, TM8 and TM10 that contribute the primary binding site. Asterisks describe positions in which the residues of LeuT were mutated to the corresponding residues in hSERT. (Wang, Hui 2013)

Table 1. The mutants are labelled as Δ5 to Δ13. Each mutant has different mutated residues. Δ13 has the most mutated residues. (Wang, Hui 2013)

A

B

C

D

       

Figure 2. Comparison of sertraline, duloxetine, desvenlafaxine and mazindol binding between the Δ5, Δ6 and Δ13 variants. (a) Structural comparison of sertraline binding between Δ13 (cyans) and Δ6 (pink) mutants. (b) Structural comparison of duloxetine binding between Δ13 ( yellow) and Δ6 ( pink) mutants. (c) Structural comparison of desvenlafaxine binding between Δ13 (grey) and Δ6 (slate) mutants. (d) Structural comparison of desvenlafaxine binding between Δ6 (orange) and Δ5 ( palegreen) mutants.

More information about LeuT can be seen in the video below.