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

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.