Abstract
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 1H 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.
Highlights
Transporters for biogenic monoamines in the neurotransmitter:sodium symporter (NSS, SLC6) family are responsible for the uptake of the neurotransmitters serotonin, dopamine, and norepinephrine into the presynaptic neuron, for the termination of synaptic transmission.[1]
The results identify the specific locations of secondary structure elements and indicate that they are interspersed with intrinsically unstructured regions
The results of the FRET analysis indicated that the V71E mutation does not have any structural effect. We used this mutant for further filtering; we modeled the V71E mutant of each model in filtering step resulted in 1724 models (F1724), performing 500 Modeller iterations per V71E side-chain replacement
Summary
Transporters for biogenic monoamines in the neurotransmitter:sodium symporter (NSS, SLC6) family are responsible for the uptake of the neurotransmitters serotonin, dopamine, and norepinephrine into the presynaptic neuron, for the termination of synaptic transmission.[1]. Using an experimentally derived relationship between Rh values for native and denatured proteins, the expected values for a globular or unfolded SERT-Nter-H8 (95 amino acids) would be 17.8 or 29.6 Å, respectively.[44] These results support the notion that the N-terminal domain contains a significant fraction of intrinsically disordered residues, alongside structured elements that are not likely to be stable β-sheets.
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