Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

Abstract

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

Binding Mode Selection Determines the Action of Ecstasy Homologs at Monoamine Transporters

“…We previously described procedures to generate free outward-facing homology models for DAT inserted into a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, which were optimized and tested for stability in 200 ns molecular dynamics simulations (Stockner et al, 2013. At the end of the simulations, the three best-scored models were selected for docking.…”

“…The protein models were created following the procedure published earlier (Stockner et al, 2013. The models were based on the outward-facing structures of the leucine transporter, LeuT Aa , crystallized from Aquifex aeolicus (Protein Data Bank identity 3F3A) (Singh et al, 2007) because the models created from the occluded conformation resulted in a potentially too narrow S1-binding site.…”

Markov State-Based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

“…They have offered an essential molecular context for the investigation of the mechanism of uphill neurotransmitter reuptake transport enabled by the coupling with the transmembrane Na + gradient 1,10 . Our current understanding of functional mechanisms in NSS has been further shaped by the large body of structure-function studies of LeuT and other related bacterial transporters, carried out both experimentally 11-17 and computationally [11][12][13][14]16,[18][19][20][21][22][23] , and more recently by findings from molecular dynamics (MD) simulations of DAT [24][25][26][27][28][29] and SERT 30,31 constructs. Together, these studies have suggested for the transport cycle in NSS proteins an allosteric process that is consistent with the alternating access mechanism 32 in which concerted dynamic rearrangements on the extracellular (EC) and intracellular (IC) sides of the transporter result from ion-and ligand-specific conformational rearrangements.…”

Conformationally Sensitive Proximity of Extracellular Loops 2 and 4 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis

“…Interestingly, our results are in harmony with earlier observations on the inhibition of the dopamine transporter DAT by Zn 2ϩ (29,30). The target of the divalent cation is an endogenous zinc binding site formed by four amino acid residues, two from EL2 and two from EL4 (31). Because the inhibition by zinc is not due to the formation of a covalent bound, it is impossible to monitor inhibition following preincubation under conditions favoring outward-or inward-facing conformations.…”