C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration

Structural Diversity of Ubiquitin E3 Ligase

The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases

Structure elements can be predicted using the contact Volume among protein residues

Previously, the structure elements of dihydrofolate reductase (DHFR) were determined using comprehen­sive Ala-insertion mutation analysis, which is assumed to be a kind of protein “building blocks.” It is hypo­thesized that our comprehension of the structure elements could lead to understanding how an amino acid sequence dictates its tertiary structure. However, the comprehensive Ala-insertion mutation analysis is a time- and cost-consuming process and only a set of the DHFR structure elements have been reported so far. Therefore, developing a computational method to predict structure elements is an urgent necessity. We focused on intramolecular residue–residue contacts to predict the structure elements. We introduced a simple and effective parameter: the overlapped contact volume (CV) among the residues and calculated the CV along the DHFR sequence using the crystal structure. Our results indicate that the CV profile can recapitulate its precipitate ratio profile, which was used to define the structure elements in the Ala-insertion mutation analysis. The CV profile allowed us to predict structure elements like the experimentally determined structure elements. The strong correlation between the CV and precipitate ratio profiles indicates the importance of the intramolecular residue–residue contact in maintaining the tertiary structure. Additionally, the CVs between the structure elements are considerably more than those between a structure element and a linker or two linkers, indicating that the structure elements play a funda­mental role in increasing the intramolecular adhesion. Thus, we propose that the structure elements can be considered a type of “building blocks” that maintain and dictate the tertiary structures of proteins

Raw diffraction images of 5-Chlorotryptamine-bound trypsin

Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. We used datasets to investigate the protocol of detecting polymorphs using Hierarchical clustering (Acta D., submitted). All diffraction data were collected at BL32XU, SPring-8, using an automated data collection system ZOO. Data were acquired from four crystals of 5-Chlorotryptamine-bound trypsin. All datasets were collected using a continuous helical scan scheme for 360º oscillation with the following experimental parameters; Beam size: 10 µm × 15 µm, Wavelength: 1.0000 Å, Total dose/crystal: 10 MGy, Detector: EIGER X 9M (DECTRIS Co. Ltd.). All crystals belonged to space group P212121 with unit cell parameters roughly corresponding to a=54.5, b=58.6, c=66.6 Å

Raw diffraction images of [NiFe]-hydrogenase maturation factor HypD from Aquifex aeolicus (C360S mutant)

HypD is one of the maturation factors of [NiFe]-hydrogenase and can form a complex with other maturation factors (Muraki et al., 2019). We used datasets to investigate the protocol of detecting polymorphs using Hierarchical clustering (Acta D., submitted). All diffraction datasets were collected at BL45XU, SPring-8, using an automated data collection system ZOO. From six crystals, datasets were collected from each one using a continuous helical scan scheme for 360º oscillation with the following experimental parameters; Beam size: 20 µm × 20 µm, Wavelength: 1.0000 Å, Total dose/crystal: 10 MGy, Detector: EIGER X 9M (DECTRIS Co. Ltd.). All crystals belonged to space group P212121 with unit cell parameters roughly corresponding to a=60.0, b=62.6, c=97.6 Å

Raw diffraction images of 4-Methoxybenzamidine-bound trypsin

Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. We used datasets to investigate the protocol of detecting polymorphs using Hierarchical clustering (Acta D., submitted). All diffraction data were collected at BL32XU, SPring-8, using an automated data collection system ZOO. Data were acquired from four crystals of 4-Methoxybenzamidine -bound trypsin. All datasets were collected using a continuous helical scan scheme for 360º oscillation with the following experimental parameters; Beam size: 10 µm × 15 µm, Wavelength: 1.0000 Å, Total dose/crystal: 10 MGy, Detector: EIGER X 9M (DECTRIS Co. Ltd.). All crystals belonged to space group P212121 with unit cell parameters roughly corresponding to a=54.6, b=58.6, c=66.7 Å