Nucleosomes, the basic units of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome is well characterized, little is known about ...structures of partially unwrapped, transient intermediates. In this study, we present nine cryo-EM structures of distinct conformations of nucleosome and subnucleosome particles. These structures show that initial DNA breathing induces conformational changes in the histone octamer, particularly in histone H3, that propagate through the nucleosome and prevent symmetrical DNA opening. Rearrangements in the H2A-H2B dimer strengthen interaction with the unwrapping DNA and promote nucleosome stability. In agreement with this, cross-linked H2A-H2B that cannot accommodate unwrapping of the DNA is not stably maintained in the nucleosome. H2A-H2B release and DNA unwrapping occur simultaneously, indicating that DNA is essential in stabilizing the dimer in the nucleosome. Our structures reveal intrinsic nucleosomal plasticity that is required for nucleosome stability and might be exploited by extrinsic protein factors.
Nucleosomes, the basic unit of chromatin, package and regulate expression of eukaryotic genomes. Nucleosomes are highly dynamic and are remodeled with the help of ATP-dependent remodeling factors. ...Yet, the mechanism of DNA translocation around the histone octamer is poorly understood. In this study, we present several nucleosome structures showing histone proteins and DNA in different organizational states. We observe that the histone octamer undergoes conformational changes that distort the overall nucleosome structure. As such, rearrangements in the histone core α-helices and DNA induce strain that distorts and moves DNA at SHL 2. Distortion of the nucleosome structure detaches histone α-helices from the DNA, leading to their rearrangement and DNA translocation. Biochemical assays show that cross-linked histone octamers are immobilized on DNA, indicating that structural changes in the octamer move DNA. This intrinsic plasticity of the nucleosome is exploited by chromatin remodelers and might be used by other chromatin machineries.
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.
Nucleosomes, the basic unit of chromatin, are repetitively spaced along DNA and regulate genome expression and maintenance. The long linear chromatin molecule is extensively condensed to fit DNA ...inside the nucleus. How distant nucleosomes interact to build tertiary chromatin structure remains elusive. In this study, we used cryo-EM to structurally characterize different states of long range nucleosome core particle (NCP) interactions. Our structures show that NCP pairs can adopt multiple conformations, but, commonly, two NCPs are oriented with the histone octamers facing each other. In this conformation, the dyad of both nucleosome core particles is facing the same direction, however, the NCPs are laterally shifted and tilted. The histone octamer surface and histone tails in trans NCP pairs remain accessible to regulatory proteins. The overall conformational flexibility of the NCP pair suggests that chromatin tertiary structure is dynamic and allows access of various chromatin modifying machineries to nucleosomes.
Approximately 30%–40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is ...densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a “magic number” effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.
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•The pyrenoid matrix is not a crystalline solid and instead behaves like a liquid•The pyrenoid is inherited primarily by fission and can also be assembled de novo•The pyrenoid undergoes a reversible phase transition during cell division•Modeling reveals a “magic number” effect that governs phase transitions
The pyrenoid, a Rubisco-containing organelle that enhances carbon fixation, mixes internally and undergoes phase transitions.
We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs ...enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a β-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.
The hetero-oligomeric chaperonin of eukarya, TRiC, is required to fold the cytoskeletal protein actin. The simpler bacterial chaperonin system, GroEL/GroES, is unable to mediate actin folding. Here, ...we use spectroscopic and structural techniques to determine how TRiC promotes the conformational progression of actin to the native state. We find that actin fails to fold spontaneously even in the absence of aggregation but populates a kinetically trapped, conformationally dynamic state. Binding of this frustrated intermediate to TRiC specifies an extended topology of actin with native-like secondary structure. In contrast, GroEL stabilizes bound actin in an unfolded state. ATP binding to TRiC effects an asymmetric conformational change in the chaperonin ring. This step induces the partial release of actin, priming it for folding upon complete release into the chaperonin cavity, mediated by ATP hydrolysis. Our results reveal how the unique features of TRiC direct the folding pathway of an obligate eukaryotic substrate.
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•Actin fails to fold spontaneously, strictly requiring TRiC chaperonin for folding•Actin binding to TRiC specifies a unique topology for productive folding•ATP binding induces an asymmetric TRiC intermediate and selective actin release•Stepwise folding on and inside TRiC allows actin to access the native state
Single-molecule studies explain why the eukaryotic chaperone complex TRiC, but not the simpler bacterial chaperonin system, is able to induce the proper folding of actin.
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.
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•Oligomerization of PABP on the poly(A) tail creates a series of consecutive arches•Pan2-Pan3 deadenylase recognizes the oligomerized state of poly(A)-bound PABP•The dimerization interface of juxtaposed PABPs creates the Pan2-Pan3 docking site•The poly(A) RNP arches are flexible and moldable by the interacting proteins
A structural view of the interplay between a deadenylase and its substrate reveals how oligomers of poly(A) binding protein are recognized by the Pan2-Pan3 deadenylase and how these RNA-bound oligomers act as rulers for poly(A) tail length
We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase ...dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F1 heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.
ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo‐tomography we show that the ...ATP synthase of mammalian mitochondria is arranged in long ∼1‐μm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17‐nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of ∼0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton‐limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance.
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