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MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na+‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity. Synopsis: Bacillus halodurans MhsT transporter is the homologue of human SLC6 transporters and mediates uptake of a broad range of hydrophobic amino acids. Here, structural and functional data provide insight into the substrate recognition mechanism of MhsT with ramifications for SLC6 transporters and the larger family of LeuT‐fold proteins. Crystal structures of six MhsT/substrate complexes in the inward‐facing conformation reveal the role of the conserved unwound region of transmembrane segment (TM) 6 in substrate recognition, promiscuity and transport.The conserved (G/A/C)ΦG motif within the unwound part of TM6 is crucial for tailoring the volume of the central substrate site, thereby differentiating between branched aliphatic and larger aromatic amino acid substrates.The interaction between Glu66 located in TM2 and the unwound part of TM6 is conserved in all known conformational states and appears as a crucial mechanistic element of SLC6 transporters. [ABSTRACT FROM AUTHOR]

For membrane proteins, ab initio modelling based on a single curve of small‐angle X‐ray scattering (SAXS) is precluded by the presence of detergent molecules bound to the hydrophobic region of the protein. MEMPROT was developed for the modelling of protein–detergent complexes based on the SAXS curve of the complex, and on an a priori representation of the detergent corona by an elliptical semi‐torus surrounding the protein. In previous studies, MEMPROT has succeeded in modelling several membrane proteins solubilized in n‐dodecyl‐β‐maltopyranoside (DDM). However, it has never been tested on proteins solubilized in other detergents. To understand whether the geometrical shape currently parametrized in MEMPROT could be applied to a broader catalogue of protein–detergent complexes, here, MEMPROT was used to model the detergent corona around the multi‐hydrophobic substrate transporter from Bacillus halodurans solubilized in four different detergents, namely DDM, n‐decyl‐β‐maltopyranoside (DM), 4‐cyclohexyl‐1‐butyl‐β‐d‐maltoside (Cymal4) and decyl‐maltose‐neopentyl‐glycol (DMNG). The study indicates a significant variation in detergent shapes, depending on the type of detergent. The modelling results suggest that the elliptical semi‐torus with a circular closure is an excellent approximation for long‐tailed detergents (DDM and DM) but leads to a slightly poorer agreement with the data for DMNG and Cymal4, which have a shorter hydrophobic tail, smaller than the half‐width of the protein hydrophobic region. Here, for the latter, it is hypothesized that a corona with a flatter closure would be a better shape descriptor. [ABSTRACT FROM AUTHOR]

The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium‐ and chloride‐coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine‐like rewarding effects and offer a promising strategy for the treatment of drug use disorders. Here, we present the 3.2 Å resolution cryo‐electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non‐competitive inhibitor AC‐4‐248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. Our findings propose mechanisms for the non‐competitive inhibition of DAT and attenuation of cocaine potency by AC‐4‐248 and provide a basis for the rational design of more efficacious atypical inhibitors. [ABSTRACT FROM AUTHOR]