G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, 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 cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins.

Phosphofructokinase-1 (PFK1) is an essential glycolysis enzyme as it catalyzes the step committing glucose to breakdown. Webb et al. show that the liver PFK1 isoform assembles into filaments in a tetramer- and substrate-dependent manner, providing insights into the spatial organization of isoform-specific glucose metabolism in cells.

• The VWF D9 domains are flexibly tethered entities projecting outside antiparallel dimers of the VWF D3 domain. • Extensive interactionsbetween the VWF D9 domain and primarily the FVIII C1 domain mediate VWF-FVIII association.Binding to the von Willebrand factor (VWF) D9D3 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 D9D3 alone and in complex with FVIII. The D9D3 dimer ([D9D3] 2 ) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D9. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D9 domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. IntroductionThe strong association of plasma factor VIII (FVIII) with circulating von Willebrand factor (VWF) secures FVIII from rapid clearance in the blood. The VWF-FVIII complex forms through a high-affinity interaction between the FVIII light chain and the VWF D9D3 domains. 1Mutations within VWF that abrogate or abolish this high-affinity binding lead to type 2N von Willebrand disease, a condition characterized by reduced plasma levels of FVIII. 2The tertiary structure of mature VWF, particularly at the N-terminal D9D3 domains, regulates the affinity for FVIII. VWF circulates as a multi-subunit protein comprising repeated domains that distinctly facilitate VWF packaging and hemostasis.3 The VWF propeptide (domains D1and D2) catalyzes the multimerization of VWF via intermolecular disulfide bonds at the D3 domain ( Figure 1A). 4 In the absence of propeptide-dependent posttranslational modifications to the D9D3 domains, VWF binds FVIII with reduced affinity.5 Cleavage of the propeptide by furin facilitates FVIII stabilization in the circulation. 6 We and others have previously reported that VWF fragments are sufficient to bind FVIII and that propeptide processing of these VWF fragments enhances the affinity for FVIII.7-9 Several of these VWF fragments were also sufficient to elevate FVIII levels in VWF-deficient mice. 7To further explore the association between VWF and FVIII, we used single-particle negative-stain electron microscopy (EM) to characterize the architecture of dimeric VWF D9D3 domains ([D9D3] 2 ) alone and in complex with FVIII. Study designProtein expression, purification, and analyses are detailed in supplemental Data available on the Blood Web site. The online version of this article contains a data supplement. Results and discussionThere is an Inside Blood Commentary on this article in this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734. , each monomer appears as an ovoid density along the dimer symmetry axis accompanied by a weaker elongated density, which we term the "handle," in the periphery. The dimensions of the handle are ;20Å ...

DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson–Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ~2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications.

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that metabolic enzymes from diverse pathways can assemble into filaments. We show that the liver isoform of glycolytic “gatekeeper” enzyme phosphofructokinase‐1 (PFKL), which catalyzes the step committing glucose to breakdown, forms filaments in vitro and dynamic punctae in cells. Recombinant PFKL, but not platelet or muscle isoforms, assembles into filaments in a concentration‐, substrate‐, and tetramer‐dependent manner. Filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer as determined by negative‐stain electron micrographs. Quantified live‐cell imaging shows PFKL‐EGFP appears as dynamic puncta that are enriched at discrete locations at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform‐specific glucose metabolism in cells. Support or Funding Information West Virginia University Start up funds