Purification and characterization of eukaryotic ATP-dependent transporters homologously expressed in Pichia pastoris for structural studies by cryo-electron microscopy.

Membrane proteins play an essential role in all living organisms. Although there have been numerous efforts in the past to elucidate the structure and function of eukaryotic primary active transporters, knowledge about the majority of these membrane proteins is still minimal. This is often due to their low availability and complex handling. In this study, we homologously expressed three ATP-dependent transport proteins, STE6-2p, NEO1-p, and YPK9-p, in Pichia pastoris and subsequently optimized the solubilization and purification processes. Sequential use of different mild detergents and utilization of hydrophilic matrices in the purification procedure allowed us to obtain all three transporters monodisperse and in high purity, enabling initial structural analysis by cryo-electron microscopy. Using the respective substrates, we determined the specific activity of all target proteins using an ATPase assay. This study opens the door to further functional and structural studies of this pharmacologically important class of membrane proteins.

The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.