Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Ligand-induced architecture of the leptin receptor signaling complex.

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.

Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII.

Binding to the von Willebrand factor (VWF) D'D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D'D3 alone and in complex with FVIII. The D'D3 dimer ([D'D3]2) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D'. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D' domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. Modeling the FVIII structure into the three-dimensional EM reconstructions of [D'D3]2-FVIII ternary and quaternary complexes indicates conformational rearrangements of the FVIII C domains compared with their disposition in the unbound state. These results illustrate the cooperative plasticity between VWF and FVIII that coordinate their high-affinity interaction.

Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human.

Histone H3 lysine 4 (H3K4) methylation is catalyzed by the highly evolutionarily conserved multiprotein complex known as Set1/COMPASS or MLL/COMPASS-like complexes from yeast to human, respectively. Here we have reconstituted fully functional yeast Set1/COMPASS and human MLL/COMPASS-like complex in vitro and have identified the minimum subunit composition required for histone H3K4 methylation. These subunits include the methyltransferase C-terminal SET domain of Set1/MLL, Cps60/Ash2L, Cps50/RbBP5, Cps30/WDR5, and Cps25/Dpy30, which are all common components of the COMPASS family from yeast to human. Three-dimensional (3D) cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and two-dimensional (2D) EM analysis of the individual subcomplexes reveal a Y-shaped architecture with Cps50 and Cps30 localizing on the top two adjacent lobes and Cps60-Cps25 forming the base at the bottom. EM analysis of the human complex reveals a striking similarity to its yeast counterpart, suggesting a common subunit organization. The SET domain of Set1 is located at the juncture of Cps50, Cps30, and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation. This structural arrangement suggested that COMPASS family members function as exo-methylases, which we have confirmed by in vitro and in vivo studies.

Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied ß(2)-adrenoceptor (ß(2)AR) in complex with the heterotrimeric G protein Gs (Gαsßγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

The Vps13p-Cdc31p complex is directly required for TGN late endosome transport and TGN homotypic fusion.

Yeast VPS13 is the founding member of a eukaryotic gene family of growing interest in cell biology and medicine. Mutations in three of four human VPS13 genes cause autosomal recessive neurodegenerative or neurodevelopmental disease, making yeast Vps13p an important structural and functional model. Using cell-free reconstitution with purified Vps13p, we show that Vps13p is directly required both for transport from the trans-Golgi network (TGN) to the late endosome/prevacuolar compartment (PVC) and for TGN homotypic fusion. Vps13p must be in complex with the small calcium-binding protein Cdc31p to be active. Single-particle electron microscopic analysis of negatively stained Vps13p indicates that this 358-kD protein is folded into a compact rod-shaped density (20 × 4 nm) with a loop structure at one end with a circular opening ∼6 nm in diameter. Vps13p exhibits ATP-stimulated binding to yeast membranes and specific interactions with phosphatidic acid and phosphorylated forms of phosphatidyl inositol at least in part through the binding affinities of conserved N- and C-terminal domains.