RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160, is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane that covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

We recently reported the development of a computational method for the design of co-assembling, multi-component protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials. This article is protected by copyright. All rights reserved.

The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (βVR). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

The chaperone Hsc70 drives the clathrin assembly-disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 A resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4-S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π-π bonds and cation-π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

A variant Hb zeta(2)beta(s)(2) that is formed from sickle hemoglobin (Hb S; alpha(2)beta(s)(2)) by exchanging adult alpha-globin with embryonic zeta-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb zeta(2)beta(s)(2) inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (alpha(2)beta(2)), Hb zeta(2)beta(s)(2) exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95 angstrom resolution crystal structure of human Hb zeta(2)beta(s)(2) that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb zeta(2)beta(s)(2) displays a central water cavity, a zeta 1-beta(s)2 (or zeta 2-beta(s)1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal beta His146 salt-bridge interactions, and a beta-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the zeta 1-beta(s1) dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb zeta(2)beta(s)(2) and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.

The human GABA receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA receptor has two subunits, GABA and GABA, that are structurally homologous but perform distinct and complementary functions. GABA recognizes orthosteric ligands, while GABA couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABA receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.