G_s protein peptidomimetics as allosteric modulators of the β₂-adrenergic receptor

A series of G(s) protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β(2)-adrenergic receptor (β(2)AR) in complex with the G(s) protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular G(s) protein binding site of the β(2)AR. Peptidomimetics were designed to mimic the 15 residue C-terminal α-helix of the G(s) protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified β(2)AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of β(2)AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native G(s) protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation

Yeast surface display platform for rapid discovery of conformationally selective nanobodies

Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research