Substrate binding and translocation of the serotonin transporter studied by docking and molecular dynamics simulations (original) (raw)
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Proteins: Structure, Function, and Bioinformatics, 2009
To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT Aa ) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT Aa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT Aa structure, matches sitedirected mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/ dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.
Structure and Regulatory Interactions of the Cytoplasmic Terminal Domains of Serotonin Transporter
Biochemistry, 2014
Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein−protein interactions involving the small cytoplasmic segments at the amino-and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1 H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the "rocking bundle" hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.
A Conformationally Sensitive Residue on the Cytoplasmic Surface of Serotonin Transporter
Journal of Biological Chemistry, 2001
Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479 -1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained ϳ32% of wild type transport activity and ϳ56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na ؉ and at 4°C, their ability to protect Cys-357 required Na ؉ and was prevented at 4°C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K ؉ ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity. . 1 The abbreviations used are: SERT, serotonin transporter; 5-HT, serotonin; DAT, dopamine transporter; NET, norepinephrine transporter; MTSEA, 2-(aminoethyl)methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; IL1 and IL3, inner loop 1 and 3, respectively; MTS, methanethiosulfonate; -CIT, 2--carbomethoxy-3--(4-[ 125 I]iodophenyl)tropane; NMDG, Nmethyl D-glucamine.
Journal of Pharmacology and Experimental Therapeutics, 2008
The human serotonin transporter (hSERT) regulates the spatial and temporal actions of serotonin (5-HT) neurotransmission by removing 5-HT from the synapse. Previous studies have identified residues in the third transmembrane helix (TMH) that may be important for substrate translocation or antagonist recognition. We identified hSERT residues in TMH III that are divergent from drosophila SERT (dSERT) and employed species-scanning mutagenesis to generate reciprocal mutants. Transport inhibition assays suggest that the potency of substituted amphetamines was decreased for the hSERT mutants A169D, I172M, and S174M. Additionally, there was a loss of potency for several antidepressants and 3-phenyltropane analogs for the I172M mutant. These results suggest that residues in TMH III may contribute to antagonist recognition. We carried out Comparative Molecular Field Analyses (CoMFA) using selectivity fields to directly visualize the mutation-induced effects of antagonist potency for antidepressants, 3-phenyltropane analogs, and amphetamines. The hSERT I172M selectivity field analysis for the 3-phenyltropane analogs revealed that electrostatic interactions resulted in decreased potency. The amphetamine and antidepressant selectivity field analyses reveal the observed decreases in potencies for the hSERT I172M mutant are due to a change in tertiary structure of the hSERT protein and are not due to disruption of a direct binding site. Finally, the hSERT mutant A169D displayed altered kinetics for sodium binding, indicating that this residue may lie near the putative sodium-binding site. A SERT homology model developed from the Aquifex aeolicus leucine transporter structure provides a structural context for further interpreting the results of the TMH III mutations.
Molecular Pharmacology, 2012
Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT 1A receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Fö rster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted con-tacts (Trp175 4.64 , Tyr198 5.41 , Arg151 4.40 , and Arg152 4.41 ) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp175 4.64 in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/ TM5 can form an interaction interface in 5-HT 1A receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.
Accessibility and conformational coupling in serotonin transporter predicted internal domains
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002
The intracellular topology of serotonin transporter (SERT) was examined using mutants containing single cysteine residues in the predicted cytoplasmic domain of the protein. Cysteine residues in each predicted cytoplasmic domain, including the NH2 and COOH termini and the five predicted internal loops, reacted with methanethiosulfonate (MTS) reagents only when the plasma membrane was permeabilized with digitonin or in membrane preparations but not in intact cells. The reaction was monitored by inactivation of high-affinity binding activity and by incorporation of biotin groups into the protein. Of the seven endogenous cysteine residues predicted to lie in the cytoplasmic domain, modification of only Cys-357 in the third internal loop (IL3) led to loss of activity. Cys-15 in the NH2 terminus and Cys-622 in the COOH terminus also reacted with MTS reagents. Modification of cysteine residues inserted at positions 137 in IL1, 277 in IL2, and 441 in IL4 also led to inactivation, and at po...
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
The intracellular topology of serotonin transporter (SERT) was examined using mutants containing single cysteine residues in the predicted cytoplasmic domain of the protein. Cysteine residues in each predicted cytoplasmic domain, including the NH2 and COOH termini and the five predicted internal loops, reacted with methanethiosulfonate (MTS) reagents only when the plasma membrane was permeabilized with digitonin or in membrane preparations but not in intact cells. The reaction was monitored by inactivation of high-affinity binding activity and by incorporation of biotin groups into the protein. Of the seven endogenous cysteine residues predicted to lie in the cytoplasmic domain, modification of only Cys-357 in the third internal loop (IL3) led to loss of activity. Cys-15 in the NH2 terminus and Cys-622 in the COOH terminus also reacted with MTS reagents. Modification of cysteine residues inserted at positions 137 in IL1, 277 in IL2, and 441 in IL4 also led to inactivation, and at po...
Ligand Effects on Cross-linking Support a Conformational Mechanism for Serotonin Transport
Journal of Biological Chemistry, 2009
Serotonin transporter (SERT) is responsible for the re-uptake of 5-hydroxytryptamine (5-HT) from the synaptic cleft after release from serotonergic neurons. We show here that cysteine residues at positions in transmembranes 1 and 3 of SERT, like the corresponding positions in the ␥-aminobutyric acid transporter, can be cross-linked using copper(II)(1,10-phenanthroline) 3 . The presence of a cross-link was detected by a novel methionine mutagenesis strategy. A change in mobility for an N-terminal cyanogen bromide fragment accompanied disulfide cross-linking of the two cysteine residues. Cross-linking also inhibited transport, and this process was blocked by cocaine, which is expected to stabilize SERT in conformations where the two positions are separated, but cocaine did not decrease accessibility of either of the two cysteines to modification by 2-aminoethyl methanethiosulfonate. Cysteine was required at both positions on the same molecule for efficient cross-linking, indicating that the reaction was intramolecular.
The Cytoplasmic Substrate Permeation Pathway of Serotonin Transporter
Journal of Biological Chemistry, 2006
Serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin (5-HT) and is a target for antidepressant drugs and psychostimulants. It is a member of a large family of neurotransmitter and amino acid transporters. A recent study using site-directed cysteine modification identified a helical region of the transporter with high accessibility to the cytoplasm. Subsequently, the high resolution structure of LeuT, a prokaryotic homologue, showed that the residues corresponding to this helical region are part of the fifth transmembrane domain. The accessibility of these positions is now shown to depend on conformational changes corresponding to interconversion of SERT between two forms that face the extracellular medium and the cytoplasm, respectively. Binding of the extracellular inhibitor cocaine decreased accessibility at these positions, whereas 5-HT, the transported substrate, increased it. The effect of 5-HT required the simultaneous presence of Na ؉ and Cl ؊ , which are transported into the cell together (symported) with 5-HT. In light of the LeuT structure, these results begin to define the pathway through which 5-HT diffuses between its binding site and the cytoplasm. They also confirm a prediction of the alternating access model for transport, namely, that all symported substrates must bind together before translocation. . 2 The abbreviations used are: SERT, serotonin transporter; 5-HT, 5-hydroxytryptamine; symport, transport together in the same direction; antiport, transport together in opposite directions; -CIT, 2-carbomethoxy-3-(4-iodophenyl)tropane; MTS, methanethiosulfonate; MTSEA, 2-(aminoethyl)methanethiosulfonate hydrobromide; MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate; X5C, SERT C15A/C21A/C109A/C357I/C622A; TM, transmembrane domain; EL, extracellular loop; NMDG, N-methyl-D-glucamine.
Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway
Journal of Biological Chemistry, 2010
Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT Aa) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln 332 in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln 332 to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.