Structural insights into the inhibition of glycine reuptake.

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

High-throughput screening of BAM inhibitors in native membrane environment.

The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin had a tenfold higher potency than in synthetic membranes, highlighting the need for native conditions in antibiotics development. The assay is successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of antibiotics targeting BAM.

Synthetic single domain antibodies for the conformational trapping of membrane proteins.

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.