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 protein–coupled receptor is predominantly activated by its cognate agonist substance P (SP) but also by the closely related neurokinins A and B. Here, we report cryo–electron 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

Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts

Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation

From DARPins to LoopDARPins: novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display

Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold,consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomol arrange against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30 pM were isolated with only a single round of ribosome display,an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications

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 Mg$^{2+}$ 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

Co-crystallization with conformation-specific designed ankyrin repeat proteins explains the conformational flexibility of BCL-W

BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85Å, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes

New views into class B GPCRs from the crystal structure of PTH1R

The parathyroid hormone 1 receptor (PTH1R) is a major regulator of mineral ion homeostasis and bone metabolism and is thus considered an attractive drug target for the treatment of disorders in calcium metabolism and bone-related diseases such as osteoporosis. PTH1R is a member of the class B of GPCRs, which all share a dynamic multidomain binding mechanism to the peptide hormone. For a long time, these complexes have been recalcitrant to structural studies despite their great therapeutic relevance. Through extensive engineering of both the receptor and the peptide agonist ligand, we were able to determine the first high-resolution structure of a PTH1R-agonist complex. Comparisons of the PTH1R crystal structure with subsequently reported cryo-electron microscopy structures of the same receptor in complex with a G protein, as well as with other class B GPCR structures bound to antagonists, reveal new insights into the two-step activation mechanism of class B GPCRs and extend our understanding of the precise molecular rearrangements during receptor activation

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

Crystal structures of the human neurokinin 1 receptor in complex with clinically used antagonists

Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK receptor (NKR) bound to two small-molecule antagonist therapeutics - aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NKR, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NKR bound to netupitant establish a structural rationale for the lack of basal activity in NKR. Taken together, these co-structures provide a comprehensive structural basis of NKR antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family