Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

The serotonergic system plays an important role in many psychiatric disorders. Its role in depression is well established (1). The majority of antidepressants, including TCAs, 6 cause increased synaptic serotonin (5-HT) levels via blockade of 5-HT reuptake into the presynaptic neuron (2-4) by competitive inhibition of hSERT. TCAs have been in clinical use since the 1950s, with imipramine being the first and most prominent compound (5). In severely depressed hospitalized patients, TCAs appear to be more efficacious than selective serotonin reuptake inhibitors (6). TCAs remain in widespread clinical use, especially for treatment-resistant depression (7).hSERT belongs to the neurotransmitter sodium symporter family (2, 8). These transporters utilize the electrochemical gradient of sodium and chloride ions to accumulate 5-HT against its own gradient (9 -11). No experimentally solved structures of the monoamine transporters exist, including hSERT and the dopamine and norepinephrine transporters. However, the structure of LeuT, a bacterial homolog of the neurotransmitter sodium symporters, in a substrate-occluded conformation, was reported in 2005 (12). Two sodium ions (12) and a chloride ion bind near the central substrate site (13-14) structurally and functionally coupling sodium and chloride binding to substrate binding. Recently, different transport mechanisms have been suggested (15,16).Subsequently, a low affinity noncompetitive binding site for TCAs in the extracellular vestibule of the LeuT 11 Å above the central binding site was identified (17,18). The relevance of the LeuT vestibular site for TCA binding to the physiologically relevant target, hSERT, is a matter of debate. This study identifies the central binding site, not the putative vestibular site, as relevant for TCA binding to hSERT and furthermore describes and validates the orientation of TCAs within this site.In this paper, we present induced fit docking studies of imipramine and selected analogs in the previously described homology models of hSERT (19). We present binding affinity studies of 10 imipramine analogs (Fig. 1 Copenhagen Ø, Denmark. 4 To whom correspondence may be addressed. E-mail: birgit@chem.au.dk. 5 To whom correspondence may be addressed. E-mail: owiborg@post.tele.dk. 6 The abbreviations used are: TCA, tricyclic antidepressant; 5-HT, serotonin; hSERT, human SERT; WT, wild type; PaMLAC, paired mutation ligand analog complementation; MD, molecular dynamics; r.m.s., root mean square.

The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of the serotonin transporter. Although recent crystal structures have identified several potential cholesterol binding sites it is unclear whether any of these potential cholesterol sites are occupieed by cholesterol and functionally relevant. In the present study we focus on the conserved Cholesterol Site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5 and TM7. By molecular dynamics simulations we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation and vice versa mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity and turn-over rates. Taken together we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.

The two enantiomers of the antidepressant citalopram inhibit the human serotonin transporter substantially differently. Previous studies revealed Tyr95 and Ile172 as important for citalopram binding, however, the overall orientation of the ligands in the binding site and the protein-ligand interaction points remain unknown. The binding of S- and R-citalopram to a human serotonin transporter homology model are herein examined via docking simulations including induced fit effects. For a better description of the formal charges of the ligand when bound inside the protein, polarization effects of the protein were included by additional quantum-polarized ligand docking calculations, where ligand charges are evaluated using QM/MM calculations. By this approach a much clearer picture emerged of the positions of the functional groups of citalopram. The two enantiomers are predicted to bind in the substrate binding pocket with opposite orientations of their aromatic groups. The predicted binding modes are experimentally validated using human wild type and 15 serotonin transporter mutants and 13 optically pure citalopram analogues. Important protein-ligand interaction points were identified validating one binding model for each enantiomer. In the validated model of the high affinity enantiomer, S-citalopram, the fluorine atom is located near Ala173 and Thr439 and the cyano group is in close proximity of Phe341; these contacts are found to be reversed for the R-enantiomer.