Cryo-EM structure of a TTYH2 cis-dimer
Ontology highlight
ABSTRACT:
SUBMITTER: Stephen G Brohawn
PROVIDER: EMPIAR-10842 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.
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Project description:Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of ?-amyloid (A?) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and A? fibrillation is extensively documented, much is still unknown about the formation of these A? aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by A?(1-42), a particularly pathogenic variant of A? peptide. Our model reveals that the individual layers of the A? fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal ?-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of A? peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length A? peptide.
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Project description:Formation of 100S ribosome dimer is generally associated with translation suppression in bacteria. Trans-acting factors ribosome modulation factor (RMF) and hibernating promoting factor (HPF) were shown to directly mediate this process in E. coli. Gram-positive S. aureus lacks an RMF homolog and the structural basis for its 100S formation was not known. Here we report the cryo-electron microscopy structure of the native 100S ribosome from S. aureus, revealing the molecular mechanism of its formation. The structure is distinct from previously reported analogs and relies on the HPF C-terminal extension forming the binding platform for the interactions between both of the small ribosomal subunits. The 100S dimer is formed through interactions between rRNA h26, h40, and protein uS2, involving conformational changes of the head as well as surface regions that could potentially prevent RNA polymerase from docking to the ribosome.Under conditions of nutrient limitation, bacterial ribosomes undergo dimerization, forming a 100S complex that is translationally inactive. Here the authors present the structural basis for formation of the 100S complexes in Gram-positive bacteria, shedding light on the mechanism of translation suppression by the ribosome-silencing factors.
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Project description:The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryoelectron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 Å. Our structure reveals that the BChE tetramer is organized as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central lamellipodin-derived oligopeptide with a proline-rich attachment domain (PRAD) sequence that adopts a polyproline II helical conformation and runs antiparallel. The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases but may provide a promising template for designing other proteins with improved circulatory residence times.
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Project description:The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners. The modification of proteins with O-linked β-N-acetylglucosamine (OGlcNAc) plays roles in regulation of numerous cellular functions while incorrect O-GlcNAcylation patterns are linked to disease. Here, the authors report a cryo-EM structure of full-length O-GlcNAc transferase (OGT), the only enzyme responsible for O-GlcNAcylation.
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2021-05-20 | GSE148235 | GEO
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