Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA

Abstract

A three-dimensional model of the human Na+/Cl--dependent y-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuTAa) as the template. Protein structure quality checks show that the resulting structure is particular suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuTAa lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuTAa with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of potential of GABA uptake inhibitors Response to Reviewers: Reviewer #2: Obviously, no PME was used for Gromacs minimization, which is not state-of-the-art.Answer to reviewer: We are very thankful for this suggestion and have repeated the procedure of minimizing the initial protein structure using PME. In addition, we have heated the system using a simulated annealing approach in order to let the protein structure adopt better to GABA as the ligand. The differences in the resulting structure compared to the original structure are very small but, of course, in the paper we report the structure modelled with PME and after simujlated annealing.Reviewer #2:I doubt that LigandFit is the only one of the docking codes mentioned which is able to cope with the sodium-carboxylate-interaction mentioned in the text. the pose of the bound leucine in the leucine transporter LeuTAa showed that LigandFit was the best software to reproduce the orientation of the leucine in the active site and especially the interaction of the Leucine carboxyl group with the sodium atom Na1." In order to select the best-suited docking software for our needs, we have done preliminary docking calculations using the X-Ray structure of LeuTaa. These calculations showed clearly that LigandFit was best suited to reproduce especially the important interaction of the carboxylate to the sodium atom (Na1). Even with modifications in parameter files suggested by support people for FlexX and GOLD, LigandFit performed better in this very special case! And, differrent to the Pallo publication we don't need to introduce the carboxy-sodium interaction as restraint for our calculations! Reviewer #2: The much more extens...

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…Accordingly, LeuTbased homology modeling is emerging as a valuable tool in studies of the NTT members, both in purely computational studies Huang and Zhan, 2007;Jørgensen et al, 2007a,b;Indarte et al, 2008;Xhaard et al, 2008;Kardos et al, 2010;Wein and Wanner, 2010) and as a complementary tool in functional studies (Dodd and Christie, 2007;Forrest et al, 2007Forrest et al, , 2008Paczkowski et al, 2007;Vandenberg et al, 2007;Zomot et al, 2007;Beuming et al, 2008;Celik et al, 2008b;Kniazeff et al, 2008;Andersen et al, 2009bAndersen et al, , 2010Kaufmann et al, 2009;Tavoulari et al, 2009;Field et al, 2010;Koldsø et al, 2010;Sinning et al, 2010) (section III). Modeling of DAT and SERT have so far received the most attention, which probably reflects the important role of these transporters as drug targets and results in generation of several three-dimensional models of human DAT (Beuming et al, , 2008Ravna, 2006;Indarte et al, 2008) and human SERT Forrest et al, 2007;Jørgensen et al, 2007a,b;Celik et al, 2008b;Forrest et al, 2008).…”

“…Modeling of DAT and SERT have so far received the most attention, which probably reflects the important role of these transporters as drug targets and results in generation of several three-dimensional models of human DAT (Beuming et al, , 2008Ravna, 2006;Indarte et al, 2008) and human SERT Forrest et al, 2007;Jørgensen et al, 2007a,b;Celik et al, 2008b;Forrest et al, 2008). However, models of NET (Paczkowski et al, 2007;Xhaard et al, 2008), GABA (Meinild et al, 2009;Kardos et al, 2010;Skovstrup et al, 2010;Wein and Wanner, 2010), and glycine transporters Edington et al, 2009) have also been generated. In general, the high degree of sequence similarity observed between the core region of LeuT and the SLC6 NTTs produce models with very similar overall structure of the inner and outer ring regions.…”

“…For the GABA transporters, several recent studies have employed LeuT-based homology modeling and ligand docking to generate models of the potential binding mode of GABA and other ligands in the S1 site (Kardos et al, 2010;Skovstrup et al, 2010;Wein and Wanner, 2010). The models display very similar orientations of GABA in the S1 site and show that the GABA transporter displays the highest degree of similarity with LeuT in terms of the substrate binding mode.…”

Amino Acid Residues Involved in the Substrate Specificity of TauT/SLC6A6 for Taurine and γ-Aminobutyric Acid

“…24) The model simulation for hGAT1 showed a hydrogen bonding between GABA and the main chain oxygen of Tyr60 that corresponds to the Asn21 of LeuT Aa . 26,27) Therefore, regarding G57E of TauT, the reduced uptakes were caused by the Glyto-Glu substitution that introduced a bulky side chain to the substrate pocket of TauT, suggesting the involvement of Gly57, putatively located in TM1, in providing an appropriate volume of the substrate pocket and in forming a hydrogen bond between the main chain oxygen atom and interacting with the amino group of taurine and the guanidino group of GABA.…”

“…24) In LeuT Aa , these TM domains include the twelve amino acid residues, Asn21, Ala22, Leu25, Gly26, Val104, Tyr108, Phe252, Phe253, Ser256, Phe259, Ser355 and Ile359, that contribute to the substrate recognition of LeuT Aa , 24) and the detailed study of LeuT Aa has contributed to updating the structure and functional residues of SLC6A. [25][26][27][28][29] Therefore, it would be helpful to use the information obtained in the study of LeuT Aa to investigate the mechanism for substrate recognition of TauT. In the present study, to investigate this mechanism, the amino acid resides of TauT were substituted with the corresponding residues of GATs, by referring to the functional residues of LeuT Aa , and the uptakes of taurine and GABA by TauT-expressing Xenopus laevis oocytes were determined.…”