Gatterdam, K. | bioRxiv Science

bioRxiv2021

Dynamic in situ confinement triggers ligand-free neuropeptide receptor signaling

Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and mesoscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a chelator nanotool. Due to the multivalent interaction, we observed a dynamic exchange in the microscale confined regions. Fast Y2R enrichment in clustered areas triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading, and migration. We revealed that the cell response to ligand-induced activation was amplified when cells were pre-clustered by the nanotool. Ligand-independent signaling by clustering differed from ligand-induced activation in the binding of arrestin-3 as downstream effector, which was recruited to the confined regions only in the presence of the ligand. This approach enables in situ clustering of membrane receptors and raises the possibility to explore different modalities of receptor activation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/472742v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@9c7823org.highwire.dtl.DTLVardef@93660corg.highwire.dtl.DTLVardef@110c414org.highwire.dtl.DTLVardef@1b906d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

The structure of HiSiaQM defines the architecture of tripartite ATP-independent periplasmic (TRAP) transporters

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria and archaea and provide important uptake routes for many metabolites 1-3. They consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains) that form a functional unit 4. While the structures of the P-domains are well-known, an experimental structure of any QM-domain has been elusive. HiSiaPQM is a TRAP transporter for the monocarboxylate sialic acid, which plays a key role in the virulence of pathogenic bacteria 5. Here, we present the first cryo-electron microscopy structure of the membrane domains of HiSiaPQM reconstituted in lipid nanodiscs. The reconstruction reveals that TRAP transporters consist of 15 transmembrane helices and are structurally related to elevator-type transporters, such as GltPh and VcINDY 6, 7. Whereas the latter proteins function as multimers, the idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. Structural and mutational analyses together with an AlphaFold 8 model of the tripartite (PQM) complex reveal the structural and conformational coupling of the substrate-binding protein to the transporter domains. Furthermore, we characterize high-affinity VHHs that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake in vivo. Thereby, they also confirm the orientation of the protein in the membrane. Our study provides the first structure of any binding-protein dependent secondary transporter and provides starting points for the development of specific inhibitors.

biochemistry↗