SARS-CoV-2 3CLPro Dihedral Angles Reveal Allosteric Signaling.

In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state-of-the-art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the protein's C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedrals angles in transmitting allosteric signals.

Allosteric competition and inhibition in AMPA receptors.

Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators are therapeutic candidates that inhibit AMPAR activation and can compete with positive modulators to control AMPAR function through unresolved mechanisms. Here we show that allosteric inhibition pushes AMPARs into a distinct state that prevents both activation and positive allosteric modulation. We used cryo-electron microscopy to capture AMPARs bound to glutamate, while a negative allosteric modulator, GYKI-52466, and positive allosteric modulator, cyclothiazide, compete for control of the AMPARs. GYKI-52466 binds in the ion channel collar and inhibits AMPARs by decoupling the ligand-binding domains from the ion channel. The rearrangement of the ligand-binding domains ruptures the cyclothiazide site, preventing positive modulation. Our data provide a framework for understanding allostery of AMPARs and for rational design of therapeutics targeting AMPARs in neurological diseases.

Railway Catenary Condition Monitoring: A Systematic Mapping of Recent Research.

In this paper, a different approach to the traditional literature review-literature systematic mapping-is adopted to summarize the progress in the recent research on railway catenary system condition monitoring in terms of aspects such as sensor categories, monitoring targets, and so forth. Importantly, the deep interconnections among these aspects are also investigated through systematic mapping. In addition, the authorship and publication trends are also examined. Compared to a traditional literature review, the literature mapping approach focuses less on the technical details of the research but reflects the research trends, and focuses in a specific field by visualizing them with the help of different plots and figures, which makes it more visually direct and comprehensible than the traditional literature review approach.

Allosteric Competition and Inhibition in AMPA Receptors.

Excitatory neurotransmission is principally mediated by AMPA-subtype ionotropic glutamate receptors (AMPARs). Dysregulation of AMPARs is the cause of many neurological disorders and how therapeutic candidates such as negative allosteric modulators inhibit AMPARs is unclear. Here, we show that non-competitive inhibition desensitizes AMPARs to activation and prevents positive allosteric modulation. We dissected the noncompetitive inhibition mechanism of action by capturing AMPARs bound to glutamate and the prototypical negative allosteric modulator, GYKI-52466, with cryo-electron microscopy. Noncompetitive inhibition by GYKI-52466, which binds in the transmembrane collar region surrounding the ion channel, negatively modulates AMPARs by decoupling glutamate binding in the ligand binding domain from the ion channel. Furthermore, during allosteric competition between negative and positive modulators, negative allosteric modulation by GKYI-52466 outcompetes positive allosteric modulators to control AMPAR function. Our data provide a new framework for understanding allostery of AMPARs and foundations for rational design of therapeutics targeting AMPARs in neurological diseases.

Machine learning-based rail corrugation recognition: a metro vehicle response and noise perspective.

Rail corrugation is a common problem in metro lines, and its efficient recognition is always an issue worth studying. To recognize the wavelength and amplitude of rail corrugation, a particle probabilistic neural network (PPNN) algorithm is developed. The PPNN is incorporated with the particle swarm optimization algorithm and the probabilistic neural network. On the basis of the above, the in-vehicle noise characteristics measured in the field are used to recognize normal rail wavelengths of 30 and 50 mm. A stepwise moving window search algorithm suitable for selecting features with a fixed order was developed to select in-vehicle noise features. Sound pressure levels at 400, 500, 630 and 800 Hz of in-vehicle noise are fed into the PPNN, and the average accuracy can reach 96.43%. The bogie acceleration characteristics calculated by the multi-body dynamics simulation model are used to recognize normal rail amplitudes of 0.1 and 0.2 mm. The bogie acceleration is decomposed by the complete ensemble empirical mode decomposition with adaptive noise, and a reconstructional signal is obtained. The energy entropy of the reconstructional signal is fed into the PPNN, and the average accuracy can reach 95.40%. This article is part of the theme issue 'Artificial intelligence in failure analysis of transportation infrastructure and materials'.

Conformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism.

The bacterial divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during division. Here, we present a model of the structure and dynamics of the core complex of the E. coli divisome, supported by a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis. We focus on the septal cell wall synthase complex formed by FtsW and FtsI, and its regulators FtsQ, FtsL, FtsB, and FtsN. The results indicate extensive interactions in four regions in the periplasmic domains of the complex. FtsQ, FtsL, and FtsB support FtsI in an extended conformation, with the FtsI transpeptidase domain lifted away from the membrane through interactions among the C-terminal domains. FtsN binds between FtsI and FtsL in a region rich in residues with superfission (activating) and dominant negative (inhibitory) mutations. Mutagenesis experiments and simulations suggest that the essential domain of FtsN links FtsI and FtsL together, potentially modulating interactions between the anchor-loop of FtsI and the putative catalytic cavity of FtsW, thus suggesting a mechanism of how FtsN activates the cell wall synthesis activities of FtsW and FtsI.

Naturally mutagenic sequence diversity in a human type II topoisomerase.

Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2ß) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in TOP2B sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing TOP2B alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2ß to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.

Phosphorylation at Ser65 modulates ubiquitin conformational dynamics.

Ubiquitin phosphorylation at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the Major and CR ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, remain unresolved. Here, we perform all-atom molecular dynamics simulations using the string method with swarms of trajectories to calculate the lowest free-energy path between these two conformers. Our analysis reveals the existence of a Bent intermediate in which the C-terminal residues of the ß5 strand shift to resemble the CR conformation, while pSer65 retains contacts resembling the Major conformation. This stable intermediate was reproduced in well-tempered metadynamics calculations but was less stable for a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modeling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent ß1 strand.

Identification of Vibration Frequencies of Railway Bridges from Train-Mounted Sensors Using Wavelet Transformation.

This article presents a novel methodology to extract the bridge frequencies from the vibrations recorded on train-mounted sensors. Continuous wavelet transform is used to distinguish the bridge frequencies from the other peaks that are visible in the Fourier amplitude spectrum of the accelerations recorded on train bogies. The efficacy of the proposed method is demonstrated through numerical case studies. For this, a detailed three-dimensional finite element model that can capture the vibration characteristics of the bridge, track, and train is created, and each component of the model is separately validated. The train model used is a three-dimensional multi-degree-of-freedom system that can simulate the pitching and rolling behavior. The train was then virtually driven over the bridge at different speeds and under varying track irregularities to evaluate the robustness of the proposed method in extracting bridge frequencies from train-mounted sensors under different conditions. The proposed methodology is shown to be capable of identifying bridge modal frequencies even for aggressive track irregularity profiles and relatively high speeds of trains.

Sodium/Iodide Symporter Metastable Intermediates Provide Insights into Conformational Transition between Principal Thermodynamic States.

The Sodium/Iodide Symporter (NIS), a 13-helix transmembrane protein found in the thyroid and other tissues, transports iodide, a required constituent of thyroid hormones T3 and T4. Despite extensive experimental information and clinical data, structural details of the intermediate microstates comprising the conformational transition of NIS between its inwardly and outwardly open states remain unresolved. We present data from a combination of enhanced sampling and transition path molecular dynamics (MD) simulations that elucidate the principal intermediate states comprising the inwardly to outwardly open transition of fully bound and apo NIS under an enforced ionic gradient. Our findings suggest that in both the absence and presence of bound physiological ions, NIS principally occupies a proximally inward to inwardly open state. When fully bound, NIS is also found to occupy a rare "inwardly occluded" state. The results of this work provide novel insight into the populations of NIS intermediates and the free energy landscape comprising the conformational transition, adding to a mechanistic understanding of NIS ion transport. Moreover, the knowledge gained from this approach can serve as a basis for studies of NIS mutants to target therapeutic interventions.

Excitatory and inhibitory D-serine binding to the NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptor's bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.

Development and characterization of functional antibodies targeting NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Finding Druggable Sites in Proteins Using TACTICS.

Structure-based drug discovery efforts require knowledge of where drug-binding sites are located on target proteins. To address the challenge of finding druggable sites, we developed a machine-learning algorithm called TACTICS (trajectory-based analysis of conformations to identify cryptic sites), which uses an ensemble of molecular structures (such as molecular dynamics simulation data) as input. First, TACTICS uses k-means clustering to select a small number of conformations that represent the overall conformational heterogeneity of the data. Then, TACTICS uses a random forest model to identify potentially bindable residues in each selected conformation, based on protein motion and geometry. Lastly, residues in possible binding pockets are scored using fragment docking. As proof-of-principle, TACTICS was applied to the analysis of simulations of the SARS-CoV-2 main protease and methyltransferase and the Yersinia pestis aryl carrier protein. Our approach recapitulates known small-molecule binding sites and predicts the locations of sites not previously observed in experimentally determined structures. The TACTICS code is available at https://github.com/Albert-Lau-Lab/tactics_protein_analysis.

Distinct axial and lateral interactions within homologous filaments dictate the signaling specificity and order of the AIM2-ASC inflammasome.

Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2PYD filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASCPYD filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently demonstrate that individual filaments assemble bidirectionally. By contrast, the recognition between AIM2 and ASC requires at least one to be oligomeric and occurs in a head-to-tail manner. Using in silico mutagenesis as a guide, we also identify specific axial and lateral interfaces that dictate the recognition and distinction between AIM2 and ASC filaments. Together, the results here provide a robust framework for delineating the signaling specificity and order of inflammasomes.

Structural biology and thermodynamics of GluD receptors.

Glutamate delta (GluD) receptors are a functionally enigmatic subfamily of ionotropic glutamate receptors. Despite sharing similar sequences and structures with AMPA, NMDA, and kainate receptors, GluD receptors do not bind glutamate nor function as ligand-gated ion channels. Binding d-serine and engaging in transsynaptic protein-protein interactions, GluD receptors are thought to undergo complex conformational rearrangements for non-ionotropic signaling that regulates synaptic plasticity. Recent structural, biochemical, and computational studies have elucidated multiple conformational and thermodynamic factors governing the unique properties of GluD receptors. Here, we review advances in biophysical insights into GluD receptors and discuss the structural thermodynamic relationships that underpin their neurobiological functions.

D-Serine Potently Drives Ligand-Binding Domain Closure in the Ionotropic Glutamate Receptor GluD2.

Despite their classification as ionotropic glutamate receptors, GluD receptors are not functional ligand-gated ion channels and do not bind glutamate. GluD2 receptors bind D-serine and coordinate transsynaptic complexes that regulate synaptic plasticity. Instead of opening the ion channel pore, mechanical tension produced from closure of GluD2 ligand-binding domains (LBDs) drives conformational rearrangements for non-ionotropic signaling. We report computed conformational free energy landscapes for the GluD2 LBD in apo and D-serine-bound states. Unexpectedly, the conformational free energy associated with GluD2 LBD closure upon D-serine binding is greater than that for AMPA, NMDA, and kainate receptor LBDs upon agonist binding. This excludes insufficient force generation as an explanation for lack of ion channel activity in GluD2 receptors and suggests that non-ionotropic conformational rearrangements do more work than pore opening. We also report free energy landscapes for GluD2 LBD harboring a neurodegenerative mutation and demonstrate selective stabilization of closed conformations in the apo state.

Crystallized Monolayer Semiconductor for Ohmic Contact Resistance, High Intrinsic Gain, and High Current Density.

The contact resistance limits the downscaling and operating range of organic field-effect transistors (OFETs). Access resistance through multilayers of molecules and the nonideal metal/semiconductor interface are two major bottlenecks preventing the lowering of the contact resistance. In this work, monolayer (1L) organic crystals and nondestructive electrodes are utilized to overcome the abovementioned challenges. High intrinsic mobility of 12.5 cm2 V-1 s-1 and Ohmic contact resistance of 40 Ω cm are achieved. Unlike the thermionic emission in common Schottky contacts, the carriers are predominantly injected by field emission. The 1L-OFETs can operate linearly from VDS  = -1 V to VDS as small as -0.1 mV. Thanks to the good pinch-off behavior brought by the monolayer semiconductor, the 1L-OFETs show high intrinsic gain at the saturation regime. At a high bias load, a maximum current density of 4.2 µA µm-1 is achieved by the only molecular layer as the active channel, with a current saturation effect being observed. In addition to the low contact resistance and high-resolution lithography, it is suggested that the thermal management of high-mobility OFETs will be the next major challenge in achieving high-speed densely integrated flexible electronics.

Understanding molecular surface doping of large bandgap organic semiconductors and overcoming the contact/access resistance in organic field-effect transistors.

The contact resistance (Rc) and the effective carrier mobility (µeff) are considered as the important indicators of the performance of organic field-effect transistors (OFETs). Conventionally, the contact resistance is regarded as the interface effect between the metal electrodes and the organic semiconductors, while the carrier mobility is correlated to the crystallinity and π-π stacking of the organic molecules. In the staggered OFETs, Rc is actually closely correlated to µeff through the channel sheet resistance. Besides, the accuracy of the carrier mobility directly extracted from the non-ideal transfer curves with significant contact effect is always questionable. Herein, a diffusion-lead surface doping approach is employed to improve the contact resistance and mobility issues simultaneously. By suppressing the trap states in the sublimated 2,7-dioctyl[1]benzothieno[3,2-b][1]benzothiophene (C8-BTBT) organic semiconductor with 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4-TCNQ), we observed a 3-fold increase in the carrier mobility from 0.5 to 1.6 cm2 V-1 s-1, and the Rc also drops remarkably from 25.7 kΩ cm to 5.2 kΩ cm. Moreover, the threshold voltage (VTH), subthreshold swing (SS) and the bias stability of the OFETs are also significantly improved. Based on the detailed characterization of the C8-BTBT film upon surface doping, including X-ray diffraction (XRD) for the film crystallinity, Kelvin probe force microscopy (KPFM) for the surface potential, trap state investigation by density of states (DOS) measurement and electrical circuit modeling for partial doping analysis, we confirmed that the spontaneous charge transfer process due to the diffusion of the F4-TCNQ dopants in the C8-BTBT matrix can lead to an effective trap filling. This technique and findings can be potentially developed into a general approach for the improvement of different performance parameters of OFETs.

Enhanced sampling of glutamate receptor ligand-binding domains.

The majority of excitatory synaptic transmission in the central nervous system is mediated by ionotropic glutamate receptors (iGluRs). These membrane-bound protein assemblies consist of modular domains that can be genetically isolated and expressed, which has resulted in a plethora of crystal structures of individual domains in different conformations bound to different ligands. These structures have presented opportunities for molecular dynamics (MD) simulation studies. To examine the free energies that govern molecular behavior, simulation strategies and algorithms have been developed, collectively called enhanced sampling methods This review focuses on the use of enhanced sampling MD simulations of isolated iGluR ligand-binding domains to characterize thermodynamic properties important to receptor function.

High Conformational Variability in the GluK2 Kainate Receptor Ligand-Binding Domain.

The kainate family of ionotropic glutamate receptors (iGluRs) mediates pre- and postsynaptic neurotransmission. Previously computed conformational potentials of mean force (PMFs) for iGluR ligand-binding domains (LBDs) revealed subtype-dependent conformational differences between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) iGluR subfamilies. Here we report PMFs for the kainate receptor GluK2 in apo and glutamate-bound states. Apo and glutamate-bound GluK2 LBDs preferentially access closed-cleft conformations. Apo GluK2 exhibits a surprisingly high degree of conformational flexibility, accessing open and closed states. Comparing across iGluR subtypes, these results are similar to glycine-binding GluN1 and GluN3A NMDA subunits and differ from glutamate-binding GluA2 and GluN2A subunits. To test the contribution of cross-lobe interactions on closed-cleft LBD stability, we computed PMFs for two GluK2 mutants, D462A and D656S. D462A, but not D656S, weakens closed-cleft conformations of the glutamate-bound LBD. Theoretical Boltzmann-weighted small-angle X-ray scattering profiles improve agreement with experimental results compared with calculations from the LBD crystal structure alone.