PT - JOURNAL ARTICLE AU - Asghar M. Razavi AU - George Khelashvili AU - Harel Weinstein TI - How structural elements added by evolution from bacterial transporters to human SLC6 homologs have enabled new functional properties AID - 10.1101/204164 DP - 2018 Jan 01 TA - bioRxiv PG - 204164 4099 - http://biorxiv.org/content/early/2018/01/30/204164.short 4100 - http://biorxiv.org/content/early/2018/01/30/204164.full AB - Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters impart an expanded set of functional properties to the eukaryotic transporters, which are not shared by the bacterial homologs that lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters, that have been suggested to involve these structural elements, are sparse. To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov State Model analysis of experimentally determined properties of the wild type and mutant hDAT constructs, which reveal a rich spectrum of interactions of the hDAT N-terminus and the mechanisms by which these contribute to regulation (e.g., by phosphorylation), or to entirely new phenotypes (e.g., reverse uptake – efflux) added in evolution. We reveal separate roles for the distal and proximal segments of the much larger N-terminus shared by the eukaryotic transporters compared to the bacterial ones, consistent with the proposal that the size of this region increased during evolution to enable more, and different, modes of regulation that are not shared with the bacterial homologs.SLC6solute carrier 6NSSneurotransmitter:sodium symporterDATdopamine transporterhDAThuman dopamine transporterSERTserotonin transporterTMtransmembraneMSMMarkov State ModeltICAtime-structure based independent component analysisTPMtransition probability matrixTPTtransition path theory