Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

Concerted structural rearrangements enable RNA channeling into the cytoplasmic Ski238-Ski7-exosome assembly.

The Ski2-Ski3-Ski8 (Ski238) helicase complex directs cytoplasmic mRNAs toward the nucleolytic exosome complex for degradation. In yeast, the interaction between Ski238 and exosome requires the adaptor protein Ski7. We determined different cryo-EM structures of the Ski238 complex depicting the transition from a rigid autoinhibited closed conformation to a flexible active open conformation in which the Ski2 helicase module has detached from the rest of Ski238. The open conformation favors the interaction of the Ski3 subunit with exosome-bound Ski7, leading to the recruitment of the exosome. In the Ski238-Ski7-exosome holocomplex, the Ski2 helicase module binds the exosome cap, enabling the RNA to traverse from the helicase through the internal exosome channel to the Rrp44 exoribonuclease. Our study pinpoints how conformational changes within the Ski238 complex regulate exosome recruitment for RNA degradation. We also reveal the remarkable conservation of helicase-exosome RNA channeling mechanisms throughout eukaryotic nuclear and cytoplasmic exosome complexes.

Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA-RNA interactions.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs from Saccharomyces cerevisiae by harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA-RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

The human SKI complex regulates channeling of ribosome-bound RNA to the exosome via an intrinsic gatekeeping mechanism.

The superkiller (SKI) complex is the cytoplasmic co-factor and regulator of the RNA-degrading exosome. In human cells, the SKI complex functions mainly in co-translational surveillance-decay pathways, and its malfunction is linked to a severe congenital disorder, the trichohepatoenteric syndrome. To obtain insights into the molecular mechanisms regulating the human SKI (hSKI) complex, we structurally characterized several of its functional states in the context of 80S ribosomes and substrate RNA. In a prehydrolytic ATP form, the hSKI complex exhibits a closed conformation with an inherent gating system that effectively traps the 80S-bound RNA into the hSKI2 helicase subunit. When active, hSKI switches to an open conformation in which the gating is released and the RNA 3' end exits the helicase. The emerging picture is that the gatekeeping mechanism and architectural remodeling of hSKI underpin a regulated RNA channeling system that is mechanistically conserved among the cytoplasmic and nuclear helicase-exosome complexes.

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding.

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

Symmetric and asymmetric receptor conformation continuum induced by a new insulin.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

CENP-C unwraps the human CENP-A nucleosome through the H2A C-terminal tail.

Centromeres are defined epigenetically by nucleosomes containing the histone H3 variant CENP-A, upon which the constitutive centromere-associated network of proteins (CCAN) is built. CENP-C is considered to be a central organizer of the CCAN. We provide new molecular insights into the structure of human CENP-A nucleosomes, in isolation and in complex with the CENP-C central region (CENP-CCR ), the main CENP-A binding module of human CENP-C. We establish that the short αN helix of CENP-A promotes DNA flexibility at the nucleosome ends, independently of the sequence it wraps. Furthermore, we show that, in vitro, two regions of human CENP-C (CENP-CCR and CENP-Cmotif ) both bind exclusively to the CENP-A nucleosome. We find CENP-CCR to bind with high affinity due to an extended hydrophobic area made up of CENP-AV532 and CENP-AV533 . Importantly, we identify two key conformational changes within the CENP-A nucleosome upon CENP-C binding. First, the loose DNA wrapping of CENP-A nucleosomes is further exacerbated, through destabilization of the H2A C-terminal tail. Second, CENP-CCR rigidifies the N-terminal tail of H4 in the conformation favoring H4K20 monomethylation, essential for a functional centromere.

Structural basis for PRC2 decoding of active histone methylation marks H3K36me2/3.

Repression of genes by Polycomb requires that PRC2 modifies their chromatin by trimethylating lysine 27 on histone H3 (H3K27me3). At transcriptionally active genes, di- and tri-methylated H3K36 inhibit PRC2. Here, the cryo-EM structure of PRC2 on dinucleosomes reveals how binding of its catalytic subunit EZH2 to nucleosomal DNA orients the H3 N-terminus via an extended network of interactions to place H3K27 into the active site. Unmodified H3K36 occupies a critical position in the EZH2-DNA interface. Mutation of H3K36 to arginine or alanine inhibits H3K27 methylation by PRC2 on nucleosomes in vitro. Accordingly, Drosophila H3K36A and H3K36R mutants show reduced levels of H3K27me3 and defective Polycomb repression of HOX genes. The relay of interactions between EZH2, the nucleosomal DNA and the H3 N-terminus therefore creates the geometry that permits allosteric inhibition of PRC2 by methylated H3K36 in transcriptionally active chromatin.

Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain.

Glucose homeostasis and growth essentially depend on the hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand-receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have resolved only site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we present the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin-site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis for a comprehensive description of ligand-receptor interactions that ultimately will inform new approaches to structure-based drug design.

Crystal structure of the archaeal A1Ao ATP synthase subunit B from Methanosarcina mazei Gö1: Implications of nucleotide-binding differences in the major A1Ao subunits A and B.

The A1Ao ATP synthase from archaea represents a class of chimeric ATPases/synthases, whose function and general structural design share characteristics both with vacuolar V1Vo ATPases and with F1Fo ATP synthases. The primary sequences of the two large polypeptides A and B, from the catalytic part, are closely related to the eukaryotic V1Vo ATPases. The chimeric nature of the A1Ao ATP synthase from the archaeon Methanosarcina mazei Gö1 was investigated in terms of nucleotide interaction. Here, we demonstrate the ability of the overexpressed A and B subunits to bind ADP and ATP by photoaffinity labeling. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to map the peptide of subunit B involved in nucleotide interaction. Nucleotide affinities in both subunits were determined by fluorescence correlation spectroscopy, indicating a weaker binding of nucleotide analogues to subunit B than to A. In addition, the nucleotide-free crystal structure of subunit B is presented at 1.5 A resolution, providing the first view of the so-called non-catalytic subunit of the A1Ao ATP synthase. Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP synthase non-catalytic alpha subunit provides new insights into the similarities and differences of these nucleotide-binding ATPase subunits in particular, and into nucleotide binding in general. The arrangement of subunit B within the intact A1Ao ATP synthase is presented.

The structure of the Pan2-Pan3 core complex reveals cross-talk between deadenylase and pseudokinase.

Pan2-Pan3 is a conserved complex involved in the shortening of mRNA poly(A) tails, the initial step in eukaryotic mRNA turnover. We show that recombinant Saccharomyces cerevisiae Pan2-Pan3 can deadenylate RNAs in vitro without needing the poly(A)-binding protein Pab1. The crystal structure of an active ~200-kDa core complex reveals that Pan2 and Pan3 interact with an unusual 1:2 stoichiometry imparted by the asymmetric nature of the Pan3 homodimer. An extended region of Pan2 wraps around Pan3 and provides a major anchoring point for complex assembly. A Pan2 module formed by the pseudoubiquitin-hydrolase and RNase domains latches onto the Pan3 pseudokinase with intertwined interactions that orient the deadenylase active site toward the A-binding site of the interacting Pan3. The molecular architecture of Pan2-Pan3 suggests how the nuclease and its pseudokinase regulator act in synergy to promote deadenylation.

VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface.

VARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer.

The binding of Varp to VAMP7 traps VAMP7 in a closed, fusogenically inactive conformation.

SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from glutamine-contributing SNAREs (Q-SNAREs) embedded in one membrane and an arginine-contributing SNARE (R-SNARE) embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp colocalizes with and binds to VAMP7, an R-SNARE that is involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7-SNARE motif is trapped between Varp and the VAMP7 longin domain, and hence Varp kinetically inhibits the ability of VAMP7 to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as VAMP7, such as Rab32-GTP.

Structural basis of the intracellular sorting of the SNARE VAMP7 by the AP3 adaptor complex.

VAMP7 is involved in the fusion of late endocytic compartments with other membranes. One possible mechanism of VAMP7 delivery to these late compartments is via the AP3 trafficking adaptor. We show that the linker of the δ-adaptin subunit of AP3 binds the VAMP7 longin domain and determines the structure of their complex. Mutation of residues on both partners abolishes the interaction in vitro and in vivo. The binding of VAMP7 to δ-adaptin requires the VAMP7 SNARE motif to be engaged in SNARE complex formation and hence AP3 must transport VAMP7 when VAMP7 is part of a cis-SNARE complex. The absence of δ-adaptin causes destabilization of the AP3 complex in mouse mocha fibroblasts and mislocalization of VAMP7. The mislocalization can be rescued by transfection with wild-type δ-adaptin but not by δ-adaptin containing mutations that abolish VAMP7 binding, despite in all cases intact AP3 being present and LAMP1 trafficking being rescued.