The LeuT-fold neurotransmitter:sodium symporter MhsT has two substrate sites

Significance The crystal structure of the human serotonin transporter (hSERT) features a drug bound not only in the primary site, but also simultaneously in a second site, thereby supporting the existence of two binding sites in neurotransmitter:sodium symporters (NSSs) as we demonstrated previously for the bacterial NSS homolog LeuT. Here, we provide evidence that MhsT, another NSS homolog, can simultaneously bind substrate molecules both in its primary substrate (S1) site and in its secondary substrate (S2) site. As in LeuT, substrate binding to both the S1 and S2 sites of MhsT is critical for transport, providing further support for a mechanistic model in which the allosteric interaction between the two substrate sites is a key element of Na + -coupled symport.