Structure and Regulatory Interactions of the Cytoplasmic Terminal Domains of Serotonin Transporter

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.

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

“…(Schicker et al, 2012). This observation is consistent with earlier reports (Just et al, 2004;Yamashita et al, 2005;Zhang and Rudnick, 2006;Forrest et al, 2008) and a computational study that used a homology model of SERT (Fenollar-Ferrer et al, 2014). The latter predicted the separation of the C and N termini upon opening of the cytoplasmic pathway.…”

Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog

“…The accessibility assay used here to measure LeuT conformational change is based on previous studies using mammalian and prokaryotic transporters in the NSS family and others (1,13,47,(73)(74)(75)(76). It has several advantages for measurements of conformational change in these proteins.…”

“…Na ϩ , in the absence of substrate amino acids, shifts the distribution between these states in favor of outwardfacing (inward-closed) forms, an effect first observed with Tyt1 (1) and subsequently with LeuT (2, 3), GABA transporter (12), and SERT (13). Addition of leucine, a poor substrate for LeuT, stabilizes an intermediate conformation likely resembling crystal structures with substrate bound in an outward-occluded conformation (3,5), whereas a good substrate, such as alanine in LeuT or tyrosine in Tyt1, overcomes the conformational restriction imposed by Na ϩ , dramatically increasing the prevalence of inward-open conformations (1,4).…”

Control of serotonin transporter phosphorylation by conformational state

“…Phosphorylation was determined under conditions that influence the distribution of SERT between inwardopen and outward-open conformations. Na + is known to stabilize SERT and other transporters in the NSS family in outward-open conformations (21)(22)(23)(24)(25). Fig.…”