Daniel Hilger

Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin-1. The structure of the β-arrestin-1–V2Rpp–Fab30 complex shows marked conformational differences in β-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the ‘lariat loop’ implicated in maintaining the inactive state of β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Structural basis for nucleotide exchange in heterotrimeric G proteins

How a receptor transmits a signal G protein–coupled receptors (GPCRs) transmit diverse external signals into the cell. When activated by an outside stimulus, they bind to a G protein inside the cell and accelerate exchange of a bound guanosine diphosphate (GDP) nucleotide for guanosine triphosphate, which initiates intercellular signaling. Dror et al. used atomic-level molecular dynamics simulations to show how GPCRs enhance GDP release. The G protein is dynamic and frequently adopts a conformation that exposes GDP even without the receptor bound. GPCR binding to this conformation favors an additional structural rearrangement that favors GDP release. The authors confirmed these predictions experimentally using double electron-electron resonance spectroscopy. Science, this issue p. 1361 Atomic-level simulations show how G protein–coupled receptors trigger G protein signaling. G protein–coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains—previously observed to separate widely upon receptor binding to expose the nucleotide-binding site—separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

Single-molecule analysis of ligand efficacy in β2AR–G-protein activation

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR–Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR–Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

Structure and dynamics of GPCR signaling complexes

G-protein-coupled receptors (GPCRs) relay numerous extracellular signals by triggering intracellular signaling through coupling with G proteins and arrestins. Recent breakthroughs in the structural determination of GPCRs and GPCR–transducer complexes represent important steps toward deciphering GPCR signal transduction at a molecular level. A full understanding of the molecular basis of GPCR-mediated signaling requires elucidation of the dynamics of receptors and their transducer complexes as well as their energy landscapes and conformational transition rates. Here, we summarize current insights into the structural plasticity of GPCR–G-protein and GPCR–arrestin complexes that underlies the regulation of the receptor’s intracellular signaling profile.In this Review, the authors discuss recent insights into the mechanism of GPCR signaling provided by structural and biophysical elucidation of receptor interactions with G proteins and arrestins.

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.Yeast surface display platform allows nanobody discovery within two to three weeks. Examples include nanobodies for crystallographic applications, targeting nonpurified antigen or conformationally selective nanobodies to two distinct human GPCRs.

Platform for rapid nanobody discovery in vitro

Camelid single-domain antibody fragments (“nanobodies”) provide the remarkable specificity of antibodies within a single immunoglobulin VHH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available.

Local membrane charge regulates β2 adrenergic receptor coupling to Gi

G protein coupled receptors (GPCRs) are transmembrane receptors that signal through heterotrimeric G proteins. Lipid modifications anchor G proteins to the plasma membrane; however, little is known about the effect of phospholipid composition on GPCR-G protein coupling. The β2 adrenergic receptor (β2AR) signals through both Gs and Gi in cardiac myocytes where studies suggest that Gi signaling may be cardioprotective. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study β2AR-Gi interactions. To investigate the role of phospholipid composition on Gs and Gi coupling, we reconstituted β2AR in detergent/lipid mixed micelles and found that negatively charged phospholipids (PS and PG) inhibit β2AR-Gi3 coupling. Replacing negatively charged lipids with neutral lipids (PC or PE) facilitated the formation of a functional β2AR-Gi3 interaction that activated Gi3. Ca2+, known to interact with negatively charged PS, facilitated β2AR-Gi3 interaction in PS. Mutational analysis suggested that Ca2+ interacts with the negatively charged EDGE motif on the carboxyl-terminal end of the αN helix of Gi3 and coordinates an EDGE-PS interaction. These results were confirmed in β2AR reconstituted into nanodisc phospholipid bilayers. β2AR-Gi3 interaction was favored in neutral lipids (PE and PC) over negatively charged lipids (PG and PS). In contrast, basal β2AR-Gs interaction was favored in negatively charged lipids over neutral lipids. In negatively-charged lipids, Ca2+ and Mg2+ facilitated β2AR-Gi3 interaction. Taken together, our observations suggest that local membrane charge modulates the interaction between β2AR and competing G protein subtypes.

Development of an antibody fragment that stabilizes GPCR/G-protein complexes.

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.The determination of high resolution structures of G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins is challenging. Here authors develop an antibody fragment, mAB16, which stabilizes GPCR/G-protein complexes and facilitates the application of high resolution cryo-EM.