Search

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid–liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid–liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid–liquid phase separation, suggesting a therapeutic route for Covid-19.One Sentence SummaryAtomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.

The protective effect of transthyretin (TTR) on cellular toxicity of β-amyloid (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aβ, a protein associated with Alzheimer's disease. TTR binds Aβ, alters its aggregation, and inhibits its toxicity both in vitro and in vivo. In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aβ oligomers. These findings suggest that the Aβ-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aβ aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aβ oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aβ catalyzed by Aβ fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease.

The protective effect of transthyretin (TTR) on cellular toxicity of amyloid-beta (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, whose pathogenic aggregation causes systemic amyloidosis. In contrast, many reports have shown that TTR binds amyloid-beta (Aβ), associated with Alzheimer’s disease, alters its aggregation, and inhibits its toxicity bothin vitroandin vivo. In this study, we question whether TTR amyloidogenic ability and its anti-amyloid inhibitory effect are associated. Our results indicate that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aβ oligomers. These findings suggest that the Aβ binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aβ aggregation and toxicity, mimicking TTR cellular protection. This segment inhibits Aβ oligomer formation and also promotes the formation of non-toxic, non-amyloid, amorphous aggregates which are more sensitive to protease digestion. This segment also inhibits seeding of Aβ catalyzed by Aβ fibrils extracted from the brain of an Alzheimer’s patient. Our results suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer’s disease.Significance statementThe pathological landmarks of Alzheimer’s disease are the formation of amyloid plaques and neurofibrillary tangles. Amyloid plaques contain fibrous structures made of aggregated amyloid-beta (Aβ). In 1982, Shirahama and colleagues observed the presence of transthyretin (TTR) in these plaques. TTR is a tetrameric protein whose aggregation causes transthyretin amyloidosis. However, TTR protects Aβ from aggregating and causing toxicity to neurons. In this study, we show that the dissociation of TTR tetramers is required to inhibit cellular toxicity caused by Aβ. In addition, we identified a minimum segment of TTR that inhibits Aβ aggregation and cellular toxicity by the formation of amorphous aggregates that are sensitive to proteases, similar to the natural effect of TTR found by others in vivo.

Propionibacterium acnes is a key therapeutic target in acne, yet this bacterium has become resistant to standard antibiotic agents. We investigated whether the human antimicrobial protein granulysin is a potential candidate for the treatment of acne. Granulysin and synthetic granulysin-derived peptides possessing a helix–loop–helix motif killed P. acnes in vitro. Modification of a helix–loop–helix peptide, 31–50, by substitution of a tryptophan for the valine at amino acid 44 (peptide 31–50v44w) to increase its interaction with bacterial surfaces also increased its antimicrobial activity. Moreover, when synthesized with d- rather than l-type amino acids, this peptide (d-31– 50v44w) became less susceptible to degradation by proteases and more effective in killing P. acnes. Granulysin peptides were bactericidal, demonstrating an advantage over standard bacteriostatic antibiotics in their control of P. acnes. Moreover, peptide d-31–50v44w killed P. acnes in isolated human microcomedone preparations. Importantly, peptides 31–50, 31–50v44w, and d-31–50v44w also have potential anti-inflammatory effects, as demonstrated by suppression of P. acnes-stimulated cytokine release. Taken together, these data suggest that granulysin peptides may be useful as topical therapeutic agents, providing alternatives to current acne therapies.

We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-Å resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules., Author Summary Vaults are large barrel-shaped particles found in the cytoplasm in all mammalian cells, which may function in innate immunity. As naturally occurring nanoscale capsules, vaults may be useful objects to engineer as delivery vehicles. In this study, we propose an atomic structure for the thin outer shell of the vault. Using x-ray diffraction and computer modeling, we have inferred a draft atomic model for the major vault protein, which forms the shell-like enclosure of the vault. The shell is made up of 96 identical protein chains, each of 873 amino acid residues, folded into 14 domains. Each chain forms an elongated stave of half the vault, as well as the cap of the barrel-like shell. Our draft atomic model is essentially an atomic-level model for the entire 9.3-MDa vault shell, which offers a guide for protein engineering to test vault functions and to exploit vault particles as nanocapsules., A draft atomic structure has been determined for the 9.3-MDa protein shell of the vault cytoplasmic particle, revealing stave-like polypeptides forming the barrel-like structure of the vault.

Backgound. The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles. Results. Crystals diffracting to 3.5a resolution have been grown of a 16-residue peptide (alpha1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha1, has previously been shown to yield crystals that diffract to 1.2a resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.

The crystal structure of the pheromone Er-1 from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-1 molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.

Phospholipase A2 (PLA2) from Indian cobra venom (Naja naja naja) was crystallized from ethanol in space group P4(3)2(1)2 in the presence of Ca2+. The x-ray crystal structure was determined to 2.3-A resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R value of 0.174 for 17,542 reflections between 6.0- and 2.3-A resolution (F > 2 sigma), including 148 water molecules. The 119-amino acid enzyme has an overall architecture strikingly similar to the other known PLA2 structures with regions implicated in catalysis showing the greatest structural conservation. Unexpectedly, three monomers were found to occupy the asymmetric unit and are oriented with their catalytic sites facing the pseudo-threefold axis with approximately 15% of the solvent accessible surface of each monomer buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms. The possible relevance of this unique trimeric structure is considered.

X-ray diffraction shows the structure of a synthetic protein model, formed from noncovalent self-association of a 12-residue peptide and of sulfate ions at low pH. This peptide is a fragment of a 16-residue polypeptide that was designed to form an amphiphilic alpha helix with a ridge of Leu residues along one helical face. By interdigitation of the leucines of four such helices, the design called for self-association into a four-alpha-helical bundle. The crystal structure (2.7 angstrom resolution; R factor = 0.215) reveals a structure more complex than the design, with both a tetramer and a hexamer. The alpha-helical tetramer with leucine interior has more oblique crossing angles than most four-alpha-helical bundles; the hexamer has a globular hydrophobic core of 12 leucine residues and three associated sulfate ions. Computational analysis suggests that the hexameric association is tighter than the tetrameric one. The consistency of the structure with the design is discussed, as well as the divergence.

Melittin is the principal protein component of bee venom and is believed to function as a lytic agent. In aqueous salt solution, it is a tetramer of identical peptides, each with 26 amino acid residues. Although its amino acid composition is unusually nonpolar, and although it is believed to integrate into membranes while lysing cells, melittin is water-soluble at neutral pH. Two crystal forms have been grown from solutions containing ammonium sulfate and sodium formate, and their x-ray diffraction patterns indicate that the melittin tetramer contains at least one 2-fold axis of rotation. Both crystal forms are suitable for high resolution x-ray structural studies. Moreover, both crystals bind several heavy atoms as judged by changes in buoyancy, so that phase determination by the method of isomorphous replacement is possible. Crystallized melittin retains its lytic activity even under the conditions of crystallization (about 70% saturated ammonium sulfate).