The N-terminal peptide of the main protease of SARS-CoV-2, targeting dimer interface, inhibits its proteolytic activity.

The main protease (Mpro) of SARS-CoV-2 cleaves 11 sites of iral polypeptide chains and generates essential non-structural proteins for viral replication. Mpro is an important drug target against COVID-19. In this study, we developed a real-time fluorometric turn-on assay system to evaluate Mpro proteolytic activity for a substrate peptide between NSP4 and NSP5. It produced reproducible and reliable results suitable for HTS inhibitor assays. Thus far, most inhibitors against Mpro target the active site for substrate binding. Mpro exists as a dimer, which is essential for its activity. We investigated the potential of the Mpro dimer interface to act as a drug target. The dimer interface is formed of domain II and domain III of each protomer, in which N-terminal ten amino acids of the domain I are bound in the middle as a sandwich. The N-terminal part provides approximately 39% of the dimer interface between two protomers. In the real-time fluorometric turn-on assay system, peptides of the N-terminal ten amino acids, N10, can inhibit the Mpro activity. The dimer interface could be a prospective drug target against Mpro. The N-terminal sequence can help develop a potential inhibitor. [BMB Reports 2023; 56(11): 606-611].

Structural Insights for ß-Lactam Antibiotics.

Antibiotic resistance has emerged as a global threat to modern healthcare systems and has nullified many commonly used antibiotics. ß-Lactam antibiotics are among the most successful and occupy approximately two-thirds of the prescription antibiotic market. They inhibit the synthesis of the peptidoglycan layer in the bacterial cell wall by mimicking the D-Ala-D-Ala in the pentapeptide crosslinking neighboring glycan chains. To date, various ß-lactam antibiotics have been developed to increase the spectrum of activity and evade drug resistance. This review emphasizes the three-dimensional structural characteristics of ß-lactam antibiotics regarding the overall scaffold, working mechanism, chemical diversity, and hydrolysis mechanism by ß-lactamases. The structural insight into various ß-lactams will provide an in-depth understanding of the antibacterial efficacy and susceptibility to drug resistance in multidrug-resistant bacteria and help to develop better ß-lactam antibiotics and inhibitors.

Comparison of Performance of Predicting the Wear Amount of Tire Tread Depending on Sensing Information.

Excessive tire wear can affect vehicle driving safety. While there are various methods for predicting the tire wear amount in real-time, it is unclear which method is the most effective in terms of the difficulty of sensing and prediction accuracy. The current study aims to develop prediction algorithms of tire wear and compare their performances. A finite element tire model was developed and validated against experimental data. Parametric tire rolling simulations were conducted using various driving and tire wear conditions to obtain tire internal accelerations. Machine-learning-based algorithms for tire wear prediction utilizing various sensing options were developed, and their performances were compared. A wheel translational and rotational speed-based (V and ω) method resulted in an average prediction error of 1.2 mm. Utilizing the internal pressure and vertical load of the tire with the V and ω improved the prediction accuracy to 0.34 mm. Acceleration-based methods resulted in an average prediction error of 0.6 mm. An algorithm using both the vehicle and tire information showed the best performance with a prediction error of 0.21 mm. When accounting for sensing cost, the V and ω-based method seems to be promising option. This finding needs to be experimentally verified.

Conformational change of organic cofactor PLP is essential for catalysis in PLP-dependent enzymes.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are ubiquitous, catalyzing various biochemical reactions of approximately 4% of all classified enzymatic activities. They transform amines and amino acids into important metabolites or signaling molecules and are important drug targets in many diseases. In the crystal structures of PLP-dependent enzymes, organic cofactor PLP showed diverse conformations depending on the catalytic step. The conformational change of PLP is essential in the catalytic mechanism. In the study, we review the sophisticated catalytic mechanism of PLP, especially in transaldimination reactions. Most drugs targeting PLP-dependent enzymes make a covalent bond to PLP with the transaldimination reaction. A detailed understanding of organic cofactor PLP will help develop a new drug against PLP-dependent enzymes. [BMB Reports 2022; 55(9): 439-446].

Frequency-Based Representation of Massive Alerts and Combination of Indicators by Heterogeneous Intrusion Detection Systems for Anomaly Detection.

Although the application of a wide range of sensors has been generalized through the development of technology, the processing of massive alerts generated through data analysis and monitoring remains a challenge. This problem is also found in cyber security because the intrusion detection system (IDS) produces a tremendous number of alerts. Massive alerts not only significantly increase resources for analysis, but also make it difficult to analyze the overall situation of the system. In order to handle massive alerts, we propose using an indicator as a frequency-based representation. The proposed indicator is generated from categorical parameters of alerts that occur within a unit time utilizing frequency and is used for situational awareness with machine learning to detect whether there is a threat or not. The advantage of using indicators is that they can determine the situation for a period without analyzing individual alerts, which helps security experts to recognize the situation in the system and focus on targets that require in-depth analysis. In addition, the conversion from the categorical parameters which is highly related to analysis to numeric parameter allows for applying machine learning. For performance evaluation, we collect data from an HAI testbed similar to real critical infrastructure and conduct experiments using indicators and XGBoost, a classification machine learning algorithm against five famous vulnerability attacks. Consequently, we show that the proposed method can detect attacks with more than 90 percent accuracy, and the performance is enhanced using heterogeneous intrusion detection systems.

Remote Plasma-Induced Synthesis of Self-Assembled MoS2/Carbon Nanowall Nanocomposites and Their Application as High-Performance Active Materials for Supercapacitors.

The objective of this study is to investigate the synthesis and influence of MoS2 on carbon nanowalls (CNWs) as supercapacitor electrodes. The synthesis of MoS2 on CNW was achieved by the introduction of hydrogen remote plasma from ammonium tetrathiomolybdate (ATTM) without deterioration of the CNWs. The topographical surface structures and electrochemical characteristics of the MoS2-CNW composite electrodes were explored using two ATTM-dispersed organic solvents-acetonitrile and dimethylformamide (DMF). In this study, CNW and MoS2 were synthesized using an electron cyclotron resonance plasma. However, hydrogen radicals, which transform ATTM into MoS2, were provided in the form of a remote plasma source. The electrochemical performances of MoS2-CNW hybrid electrodes with various morphologies-depending on the solvent and ATTM concentration-were evaluated using a three-electrode system. The results revealed that the morphology of the synthesized MoS2 was influenced by the organic solvent used and affected both the electrochemical performance and topographical characteristics. Notably, considerable enhancement of the specific capacitance was observed for the MoS2 with open top edges synthesized from DMF. These encouraging results may motivate additional research on hybrid supercapacitor electrodes and the rapid synthesis of MoS2 and other transition metal dichalcogenides.

Characteristics of Plasma Flow for Microwave Plasma Assisted Aerosol Deposition.

To validate the possibility of the developed microwave plasma source with a novel structure for plasma aerosol deposition, the characteristics of the plasma flow velocity generated from the microwave plasma source were investigated by a Mach probe with pressure variation. Simulation with the turbulent model was introduced to deduce calibration factor of the Mach probe and to compare experimental measurements for analyses of collisional plasma conditions. The results show calibration factor does not seem to be a constant parameter and highly dependent on the collision parameter. The measured plasma flow velocity, which witnessed fluctuations produced by a shock flow, was between 400 and 700 m/s. The optimized conditions for microwave plasma assisted aerosol deposition were derived by the results obtained from analyses of the parameters of microwave plasma jet. Under the optimized conditions, Y2O3 coatings deposited on an aluminum substrate were investigated using scanning electron microscope. The results presented in this study show the microwave plasma assisted aerosol deposition with the developed microwave plasma source is highly feasible for thick films with >50 µm.

Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-ß-Lactamases.

Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central ß-sheets in the conserved αß/ßα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of ß-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of ß-lactam-bound MBLs show that the binding of ß-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides ß-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

Fructuronate-tagaturonate epimerase UxaE from Cohnella laeviribosi has a versatile TIM-barrel scaffold suitable for a sugar metabolizing biocatalyst.

Xylan and pectin are major structural components of plant cell walls. There are two independent catabolic pathways for xylan and pectin. UxaE bridges these two pathways by reversibly epimerizing D-fructuronate and D-tagaturonate. The crystal structure of UxaE from Cohnella laeviribosi (ClUxaE) shows a core scaffold of TIM-barrel with a position-changing divalent metal cofactor. ClUxaE has the flexible metal-coordination loop to allow the metal shift and the extra domains to bind a phosphate ion in the active site, which are important for catalysis and substrate specificity. Elucidation of the structure and mechanism of ClUxaE will assist in understanding the catalytic mechanism of UxaE family members, which are useful for processing both xylan and pectin-derived carbohydrates for practical and industrial purposes, including the transformation of agricultural wastes into numerous valuable products.

Structural Study of Metal Binding and Coordination in Ancient Metallo-ß-Lactamase PNGM-1 Variants.

The increasing incidence of community- and hospital-acquired infections with multidrug-resistant (MDR) bacteria poses a critical threat to public health and the healthcare system. Although ß-lactam antibiotics are effective against most bacterial infections, some bacteria are resistant to ß-lactam antibiotics by producing ß-lactamases. Among ß-lactamases, metallo-ß-lactamases (MBLs) are especially worrisome as only a few inhibitors have been developed against them. In MBLs, the metal ions play an important role as they coordinate a catalytic water molecule that hydrolyzes ß-lactam rings. We determined the crystal structures of different variants of PNGM-1, an ancient MBL with additional tRNase Z activity. The variants were generated by site-directed mutagenesis targeting metal-coordinating residues. In PNGM-1, both zinc ions are coordinated by six coordination partners in an octahedral geometry, and the zinc-centered octahedrons share a common face. Structures of the PNGM-1 variants confirm that the substitution of a metal-coordinating residue causes the loss of metal binding and ß-lactamase activity. Compared with PNGM-1, subclass B3 MBLs lack one metal-coordinating residue, leading to a shift in the metal-coordination geometry from an octahedral to tetrahedral geometry. Our results imply that a subtle change in the metal-binding site of MBLs can markedly change their metal-coordination geometry and catalytic activity.

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5'-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.