Cryo-EM structure of the dopamine transporter with a novel atypical non-competitive inhibitor bound to the orthosteric site.

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.

Temperature-induced aggregation behavior in bovine pancreas trypsin solutions.

In this study, we investigate the effect of temperature treatment on Bovine Pancreas Trypsin (BPT) in aqueous solutions using dynamic, static and electrophoretic light scattering, fluorescence spectroscopy and circular dichroism. Static and dynamic light scattering at various solution conditions i.e. different salt content and pH, reveals that BPT aggregation is enhanced as temperature increases in a non-reversible manner. At acidic pH protein monomers are the dominant population over aggregates of globules, nevertheless the two populations co-exist at neutral and basic pH. The surface charge of the aggregates is intensified by aggregation and it is dominated by the negative residues of the protein at all pH conditions. Protein unfolding upon thermal treatment is probed by variation of the fluorescence spectrum which is caused by the exposure of tryptophan to the aqueous environment. The exposure of the hydrophobic interior of BPT upon heating may be considered as the reason of aggregation at the molecular level. Τhis study provides information that can be useful for utilizing thermal treatment protocols of BPT towards manufacturing protein-based nano formulated drugs.

Variations on a theme: crystal forms of the amino-acid transporter MhsT.

The bacterial amino-acid transporter MhsT from the SLC6A family has been crystallized in complex with different substrates in order to understand the determinants of the substrate specificity of the transporter. Surprisingly, crystals of the different MhsT-substrate complexes showed interrelated but different crystal-packing arrangements. Space-group assignment and structure determination of these different crystal forms present challenging combinations of pseudosymmetry, twinning and translational noncrystallographic symmetry.