ALS mutations in the TIA-1 prion-like domain trigger highly condensed pathogenic structures

筋萎縮性側索硬化症（ALS）の発症機構の一端を解明 --タンパク質の高密度な凝縮構造が鍵--. 京都大学プレスリリース. 2022-09-13.T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity

Quantitative comparison of zero-loss and conventional electron diffraction from two-dimensional and thin three-dimensional protein crystals.

The scattering cross-section of atoms in biological macromolecules for both elastically and inelastically scattered electrons is approximately 100,000 times larger than that for x-ray. Therefore, much smaller (<1 microm) and thinner (<0.01 microm) protein crystals than those used for x-ray crystallography can be used to analyze the molecular structures by electron crystallography. But, inelastic scattering is a serious problem. We examined electron diffraction data from thin three-dimensional (3-D) crystals (600-750 A thick) and two-dimensional (2-D) crystals (approximately 60 A thick), both at 93 K, with an energy filtering electron microscope operated at an accelerating voltage of 200 kV. Removal of inelastically scattered electrons significantly improved intensity data statistics and R(Friedel) factor in every resolution range up to 3-A resolution. The effect of energy filtering was more prominent for thicker crystals but was significant even for thin crystals. These filtered data sets showed better intensity statistics even in comparison with data sets collected at 4 K and an accelerating voltage of 300 kV without energy filtering. Thus, the energy filter will be an effective and important tool in the structure analysis of thin 3-D and 2-D crystals, particularly when data are collected at high tilt angle

Protein and Organic-Molecular Crystallography With 300kV Electrons on a Direct Electron Detector

Electron 3D crystallography can reveal the atomic structure from undersized crystals of various samples owing to the strong scattering power of electrons. Here, a direct electron detector DE64 was tested for small and thin crystals of protein and an organic molecule using a JEOL CRYO ARM 300 electron microscope. The microscope is equipped with a cold-field emission gun operated at an accelerating voltage of 300 kV, quad condenser lenses for parallel illumination, an in-column energy filter, and a stable rotational goniometer stage. Rotational diffraction data were collected in an unsupervised manner from crystals of a heme-binding enzyme catalase and a representative organic semiconductor material Ph-BTBT-C10. The structures were determined by molecular replacement for catalase and by the direct method for Ph-BTBT-C10. The analyses demonstrate that the system works well for electron 3D crystallography of these molecules with less damaging, a smaller point spread, and less noise than using the conventional scintillator-coupled camera

Ionic scattering factors of atoms that compose biological molecules

Ionic scattering factors of atoms that compose biological molecules have been computed by the multi-configuration Dirac–Fock method. These ions are chemically unstable and their scattering factors had not been reported except for O−. Yet these factors are required for the estimation of partial charges in protein molecules and nucleic acids. The electron scattering factors of these ions are particularly important as the electron scattering curves vary considerably between neutral and charged atoms in the spatial-resolution range explored in structural biology. The calculated X-ray and electron scattering factors have then been parameterized for the major scattering curve models used in X-ray and electron protein crystallography and single-particle cryo-EM. The X-ray and electron scattering factors and the fitting parameters are presented for future reference

Enantioselectivity of discretized helical supramolecule consisting of achiral cobalt phthalocyanines via chiral-induced spin selectivity effect

Abstract Enantioselectivity of helical aggregation is conventionally directed either by its homochiral ingredients or by introduction of chiral catalysis. The fundamental question, then, is whether helical aggregation that consists only of achiral components can obtain enantioselectivity in the absence of chiral catalysis. Here, by exploiting enantiospecific interaction due to chiral-induced spin selectivity (CISS) that has been known to work to enantio-separate a racemic mixture of chiral molecules, we demonstrate the enantioselectivity in the assembly of mesoscale helical supramolecules consisting of achiral cobalt phthalocyanines. The helical nature in our supramolecules is revealed to be mesoscopically incorporated by dislocation-induced discretized twists, unlike the case of chiral molecules whose chirality are determined microscopically by chemical bond. The relevance of CISS effect in the discretized helical supramolecules is further confirmed by the appearance of spin-polarized current through the system. These observations mean that the application of CISS-based enantioselectivity is no longer limited to systems with microscopic chirality but is expanded to the one with mesoscopic chirality

Crystal structure of the Hfq and catalase HPII complex.

<p>(A) Stereo diagram showing the crystal packing of the complex composed of Hfq hexamers in cyan and HPII tetramers in violet. All the four bound Hfq hexamers are displayed for the HPII tetramer at the center, whereas only two hexamers are displayed for each surrounding HPII tetramer for clarity. (B) Structure of one HPII tetramer with four bound Hfq hexamers showing interaction through their distal surfaces. Viewed in stereo. Subunits of one Hfq hexamer are displayed in cyan and green. Numbers 2 – 5 indicate the subunit number in the Hfq hexamer as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone-0078216-g004" target="_blank">Fig. 4</a>. One molecule of the HPII tetramer is displayed in tan (the C-terminal lobe) and in magenta (the other parts). A space-filling model in blue represents heme. Other models are in grey.</p

Post-Transcriptional Regulator Hfq Binds Catalase HPII: Crystal Structure of the Complex

<div><p>We report a crystal structure of Hfq and catalase HPII from <i>Escherichia coli</i>. The post-transcriptional regulator Hfq plays a key role in the survival of bacteria under stress. A small non-coding RNA (sRNA) DsrA is required for translation of the stationary phase sigma factor RpoS, which is the central regulator of the general stress response. Hfq facilitates efficient translation of <i>rpoS</i> mRNA, which encodes RpoS. Hfq helps in the function of other specific proteins involved in RNA processing, indicating its versatility in the cell. However, structural information regarding its interactions with partners is missing. Here we obtained crystals of Hfq and HPII complexes from cell lysates following attempts to overexpress a foreign membrane protein. HPII is one of two catalases in <i>E. coli</i> and its mRNA is transcribed by an RNA polymerase holoenzyme containing RpoS, which in turn is under positive control of small non-coding RNAs and of the RNA chaperone Hfq. This sigma factor is known to have a pronounced effect on the expression of HPII. The crystal structure reveals that a Hfq hexamer binds each subunit of a HPII tetramer. Each subunit of the Hfq hexamer exhibits a unique binding mode with HPII. The hexamer of Hfq interacts via its distal surface. The proximal and distal surfaces are known to specifically bind different sRNAs, and binding of HPII could affect Hfq function. Hfq-HPII complexation has no effect on catalase HPII activity.</p></div

Interactions between Hfq subunit 5 and HPII.

<p>Subunit 5 is displayed in cyan and one molecule of the HPII tetramer is color-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone-0078216-g004" target="_blank">Fig. 4A</a>. Other models are displayed in grey. Interacting residues are drawn in ball-and-stick representation overlaid with 2 Fo - Fc maps of 1.0 σ, and atoms are color-coded as: nitrogen, blue; oxygen, red; and carbon, yellow in Hfq and magenta in HPII. Bonds are depicted as black dashes. Interactions of subunits 2 and 6 with HPII are shown in Fig. S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone.0078216.s001" target="_blank">File S1</a> and those of subunits 1 and 4 are in Fig. S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone.0078216.s001" target="_blank">File S1</a>. See also Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone.0078216.s001" target="_blank">File S1</a> for bond length and type.</p

Some structural details.

<p>(A) Two HPII molecules and their interaction partners, two Hfq hexamers. Hfq subunits are color-coded as in Figs. 3B and 4B, and HPII is displayed in tan (the C-terminal lobe) and in violet (the other parts) with heme in blue. “P” and “D” denote the proximal and distal sides of the Hfq hexamer, respectively. (B) Hfq hexamer viewed from the distal side. Residues for binding to HPII are drawn in space-filling representation (see also Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone.0078216.s001" target="_blank">File S1</a>) with the single-letter amino acid code for Tyr 25 and Asn 28 in subunit 5 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078216#pone-0078216-g005" target="_blank">Fig. 5</a>). Atoms are color-coded as: carbon, yellow; nitrogen, blue; and oxygen, red. “α1” denotes the N-terminal α-helix and “β1” – “β5” β-strands. Numbers 1 - 6 in A and B indicate the subunit number in the Hfq hexamer. Only the Hfq subunits on the front side have the number in A for clarity.</p