Structural basis for the activation and ligand recognition of the human oxytocin receptor.

The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.

Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP.

Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.

Structures of neurokinin 1 receptor in complex with Gq and Gs proteins reveal substance P binding mode and unique activation features.

The neurokinin 1 receptor (NK1R) is involved in inflammation and pain transmission. This pathophysiologically important G protein­coupled receptor is predominantly activated by its cognate agonist substance P (SP) but also by the closely related neurokinins A and B. Here, we report cryo­electron microscopy structures of SP-bound NK1R in complex with its primary downstream signal mediators, Gq and Gs. Our structures reveal how a polar network at the extracellular, solvent-exposed receptor surface shapes the orthosteric pocket and that NK1R adopts a noncanonical active-state conformation with an interface for G protein binding, which is distinct from previously reported structures. Detailed comparisons with antagonist-bound NK1R crystal structures reveal that insurmountable antagonists induce a distinct and long-lasting receptor conformation that sterically blocks SP binding. Together, our structures provide important structural insights into ligand and G protein promiscuity, the lack of basal signaling, and agonist- and antagonist-induced conformations in the neurokinin receptor family.

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies.

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.

Directed evolution for high functional production and stability of a challenging G protein-coupled receptor.

Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important receptors have to be engineered to overcome their poor intrinsic stability and low expression levels. However, those GPCRs with especially poor properties cannot be successfully optimised even with the current technologies. Here, we present an engineering strategy, based on the combination of three previously developed directed evolution methods, to improve the properties of particularly challenging GPCRs. Application of this novel combination approach enabled the successful selection for improved and crystallisable variants of the human oxytocin receptor, a GPCR with particularly low intrinsic production levels. To analyse the selection results and, in particular, compare the mutations enriched in different hosts, we developed a Next-Generation Sequencing (NGS) strategy that combines long reads, covering the whole receptor, with exceptionally low error rates. This study thus gave insight into the evolution pressure on the same membrane protein in prokaryotes and eukaryotes. Our long-read NGS strategy provides a general methodology for the highly accurate analysis of libraries of point mutants during directed evolution.

Crystal structure of the human oxytocin receptor.

The peptide hormone oxytocin modulates socioemotional behavior and sexual reproduction via the centrally expressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a nonpeptidic antagonist developed as an oral drug for the prevention of preterm labor. Our structure reveals insights into the detailed interactions between the G protein-coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helices IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Furthermore, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg2+ that acts as potent, positive allosteric modulator for agonist binding. Together, these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics.