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Mutations in SARS-CoV-2 have shown effective evasion of population immunity and increased affinity to the cellular receptor angiotensin-converting enzyme 2 (ACE2). However, in the dynamic environment of the respiratory tract, forces act on the binding partners, which raises the question of whether not only affinity but also force stability of the SARS-CoV-2-ACE2 interaction might be a selection factor for mutations. Using magnetic tweezers, we investigate the impact of amino acid substitutions in variants of concern (Alpha, Beta, Gamma and Delta) and on force-stability and bond kinetic of the receptor-binding domain-ACE2 interface at a single-molecule resolution. We find a higher affinity for all of the variants of concern (>fivefold) compared with the wild type. In contrast, Alpha is the only variant of concern that shows higher force stability (by 17%) compared with the wild type. Using molecular dynamics simulations, we rationalize the mechanistic molecular origins of this increase in force stability. Our study emphasizes the diversity of contributions to the transmissibility of variants and establishes force stability as one of the several factors for fitness. Understanding fitness advantages opens the possibility for the prediction of probable mutations, allowing a rapid adjustment of therapeutics, vaccines and intervention measures., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Molecular de-extinction could offer avenues for drug discovery by reintroducing bioactive molecules that are no longer encoded by extant organisms. To prospect for antimicrobial peptides encrypted within extinct and extant human proteins, we introduce the panCleave random forest model for proteome-wide cleavage site prediction. Our model outperformed multiple protease-specific cleavage site classifiers for three modern human caspases, despite its pan-protease design. Antimicrobial activity was observed in vitro for modern and archaic protein fragments identified with panCleave. Lead peptides showed resistance to proteolysis and exhibited variable membrane permeabilization. Additionally, representative modern and archaic protein fragments showed anti-infective efficacy against A. baumannii in both a skin abscess infection model and a preclinical murine thigh infection model. These results suggest that machine-learning-based encrypted peptide prospection can identify stable, nontoxic peptide antibiotics. Moreover, we establish molecular de-extinction through paleoproteome mining as a framework for antibacterial drug discovery., Competing Interests: Declaration of interests C.F.N. provides consulting services to Invaio Sciences and is a member of the Scientific Advisory Boards of Nowture S.L. and Phare Bio. The de la Fuente Lab has received research funding or in-kind donations from United Therapeutics, Strata Manufacturing PJSC, and Procter & Gamble, none of which were used in support of this work. C.F.N. is on the Advisory Board of Cell Reports Physical Science. An invention disclosure associated with this work has been filed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Over a century ago, physicists started broadly relying on theoretical models to guide new experiments. Soon thereafter, chemists began doing the same. Now, biological research enters a new era when experiment and theory walk hand in hand. Novel software and specialized hardware became essential to understand experimental data and propose new models. In fact, current petascale computing resources already allow researchers to reach unprecedented levels of simulation throughput to connect in silico and in vitro experiments. The reduction in cost and improved access allowed a large number of research groups to adopt supercomputing resources and techniques. Here, we outline how large-scale computing has evolved to expand decades-old research, spark new research efforts, and continuously connect simulation and observation. For instance, multiple publicly and privately funded groups have dedicated extensive resources to develop artificial intelligence tools for computational biophysics, from accelerating quantum chemistry calculations to proposing protein structure models. Moreover, advances in computer hardware have accelerated data processing from single-molecule experimental observations and simulations of chemical reactions occurring throughout entire cells. The combination of software and hardware has opened the way for exascale computing and the production of the first public exascale supercomputer, Frontier, inaugurated by the Oak Ridge National Laboratory in 2022. Ultimately, the popularization and development of computational techniques and the training of researchers to use them will only accelerate the diversification of tools and learning resources for future generations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The unbinding pathway of a protein complex can vary significantly depending on biochemical and mechanical factors. Under mechanical stress, a complex may dissociate through a mechanism different from that used in simple thermal dissociation, leading to different dissociation rates under shear force and thermal dissociation. This is a well-known phenomenon studied in biomechanics whose molecular and atomic details are still elusive. A particularly interesting case is the complex formed by bacterial adhesins with their human peptide target. These protein interactions have a force resilience equivalent to those of covalent bonds, an order of magnitude stronger than the widely used streptavidin:biotin complex, while having an ordinary affinity, much lower than that of streptavidin:biotin. Here, in an in silico single-molecule force spectroscopy approach, we use molecular dynamics simulations to investigate the dissociation mechanism of adhesin/peptide complexes. We show how the Staphylococcus epidermidis adhesin SdrG uses a catch-bond mechanism to increase complex stability with increasing mechanical stress. While allowing for thermal dissociation in a low-force regime, an entirely different mechanical dissociation path emerges in a high-force regime, revealing an intricate mechanism that does not depend on the peptide's amino acid sequence. Using a dynamic network analysis approach, we identified key amino acid contacts that describe the mechanics of this complex, revealing differences in dynamics that hinder thermal dissociation and establish the mechanical dissociation path. We then validate the information content of the selected amino acid contacts using their dynamics to successfully predict the rupture forces for this complex through a machine learning model.

Human genomic diversity has been shaped by both ancient and ongoing challenges from viruses. The current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating impact on population health. However, genetic diversity and evolutionary forces impacting host genes related to SARS-CoV-2 infection are not well understood. We investigated global patterns of genetic variation and signatures of natural selection at host genes relevant to SARS-CoV-2 infection (angiotensin converting enzyme 2 [ACE2], transmembrane protease serine 2 [TMPRSS2], dipeptidyl peptidase 4 [DPP4], and lymphocyte antigen 6 complex locus E [LY6E]). We analyzed data from 2,012 ethnically diverse Africans and 15,977 individuals of European and African ancestry with electronic health records and integrated with global data from the 1000 Genomes Project. At ACE2, we identified 41 nonsynonymous variants that were rare in most populations, several of which impact protein function. However, three nonsynonymous variants (rs138390800, rs147311723, and rs145437639) were common among central African hunter-gatherers from Cameroon (minor allele frequency 0.083 to 0.164) and are on haplotypes that exhibit signatures of positive selection. We identify signatures of selection impacting variation at regulatory regions influencing ACE2 expression in multiple African populations. At TMPRSS2, we identified 13 amino acid changes that are adaptive and specific to the human lineage compared with the chimpanzee genome. Genetic variants that are targets of natural selection are associated with clinical phenotypes common in patients with COVID-19. Our study provides insights into global variation at host genes related to SARS-CoV-2 infection, which have been shaped by natural selection in some populations, possibly due to prior viral infections.

SignificanceIn the dynamic environment of the airways, where SARS-CoV-2 infections are initiated by binding to human host receptor ACE2, mechanical stability of the viral attachment is a crucial fitness advantage. Using single-molecule force spectroscopy techniques, we mimic the effect of coughing and sneezing, thereby testing the force stability of SARS-CoV-2 RBD:ACE2 interaction under physiological conditions. Our results reveal a higher force stability of SARS-CoV-2 binding to ACE2 compared to SARS-CoV-1, causing a possible fitness advantage. Our assay is sensitive to blocking agents preventing RBD:ACE2 bond formation. It will thus provide a powerful approach to investigate the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The emergence of drug-resistant bacteria calls for the discovery of new antibiotics. Yet, for decades, traditional discovery strategies have not yielded new classes of antimicrobial. Here, by mining the human proteome via an algorithm that relies on the sequence length, net charge, average hydrophobicity and other physicochemical properties of antimicrobial peptides, we report the identification of 2,603 encrypted peptide antibiotics that are encoded in proteins with biological function unrelated to the immune system. We show that the encrypted peptides kill pathogenic bacteria by targeting their membrane, modulate gut and skin commensals, do not readily select for bacterial resistance, and possess anti-infective activity in skin abscess and thigh infection mouse models. We also show, in vitro and in the two mouse models of infection, that encrypted antibiotic peptides from the same biogeographical area display synergistic antimicrobial activity. Our algorithmic strategy allows for the rapid mining of proteomic data and opens up new routes for the discovery of candidate antibiotics., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

By targeting invasive organisms, antibiotics insert themselves into the ancient struggle of the host-pathogen evolutionary arms race. As pathogens evolve tactics for evading antibiotics, therapies decline in efficacy and must be replaced, distinguishing antibiotics from most other forms of drug development. Together with a slow and expensive antibiotic development pipeline, the proliferation of drug-resistant pathogens drives urgent interest in computational methods that promise to expedite candidate discovery. Strides in artificial intelligence (AI) have encouraged its application to multiple dimensions of computer-aided drug design, with increasing application to antibiotic discovery. This review describes AI-facilitated advances in the discovery of both small molecule antibiotics and antimicrobial peptides. Beyond the essential prediction of antimicrobial activity, emphasis is also given to antimicrobial compound representation, determination of drug-likeness traits, antimicrobial resistance, and de novo molecular design. Given the urgency of the antimicrobial resistance crisis, we analyze uptake of open science best practices in AI-driven antibiotic discovery and argue for openness and reproducibility as a means of accelerating preclinical research. Finally, trends in the literature and areas for future inquiry are discussed, as artificially intelligent enhancements to drug discovery at large offer many opportunities for future applications in antibiotic development., (© 2021. The Author(s).)

Molecular interactions are essential for regulation of cellular processes from the formation of multi-protein complexes to the allosteric activation of enzymes. Identifying the essential residues and molecular features that regulate such interactions is paramount for understanding the biochemical process in question, allowing for suppression of a reaction through drug interventions or optimization of a chemical process using bioengineered molecules. In order to identify important residues and information pathways within molecular complexes, the dynamical network analysis method was developed and has since been broadly applied in the literature. However, in the dawn of exascale computing, this method is frequently limited to relatively small biomolecular systems. In this work, we provide an evolution of the method, application, and interface. All data processing and analysis are conducted through Jupyter notebooks, providing automatic detection of important solvent and ion residues, an optimized and parallel generalized correlation implementation that is linear with respect to the number of nodes in the system, and subsequent community clustering, calculation of betweenness of contacts, and determination of optimal paths. Using the popular visualization program visual molecular dynamics (VMD), high-quality renderings of the networks over the biomolecular structures can be produced. Our new implementation was employed to investigate three different systems, with up to 2.5M atoms, namely, the OMP-decarboxylase, the leucyl-tRNA synthetase complexed with its cognate tRNA and adenylate, and respiratory complex I in a membrane environment. Our enhanced and updated protocol provides the community with an intuitive and interactive interface, which can be easily applied to large macromolecular complexes.

NAMDis a molecular dynamics program designed for high-performance simulations of very large biological objects on CPU- and GPU-based architectures. NAMD offers scalable performance on petascale parallel supercomputers consisting of hundreds of thousands of cores, as well as on inexpensive commodity clusters commonly found in academic environments. It is written in C++ and leans on Charm++ parallel objects for optimal performance on low-latency architectures. NAMD is a versatile, multipurpose code that gathers state-of-the-art algorithms to carry out simulations in apt thermodynamic ensembles, using the widely popular CHARMM, AMBER, OPLS, and GROMOS biomolecular force fields. Here, we review the main features of NAMD that allow both equilibrium and enhanced-sampling molecular dynamics simulations with numerical efficiency. We describe the underlying concepts utilized by NAMD and their implementation, most notably for handling long-range electrostatics; controlling the temperature, pressure, and pH; applying external potentials on tailored grids; leveraging massively parallel resources in multiple-copy simulations; and hybrid quantum-mechanical/molecular-mechanical descriptions. We detail the variety of options offered by NAMD for enhanced-sampling simulations aimed at determining free-energy differences of either alchemical or geometrical transformations and outline their applicability to specific problems. Last, we discuss the roadmap for the development of NAMD and our current efforts toward achieving optimal performance on GPU-based architectures, for pushing back the limitations that have prevented biologically realistic billion-atom objects to be fruitfully simulated, and for making large-scale simulations less expensive and easier to set up, run, and analyze. NAMD is distributed free of charge with its source code at www.ks.uiuc.edu.

JCVI-syn3A is a minimal bacterial cell with a 543 kbp genome consisting of 493 genes. For this slow growing minimal cell with a 105 min doubling time, we recently established the essential metabolism including the transport of required nutrients from the environment, the gene map, and genome-wide proteomics. Of the 452 protein-coding genes, 143 are assigned to metabolism and 212 are assigned to genetic information processing. Using genome-wide proteomics and experimentally measured kinetic parameters from the literature we present here kinetic models for the genetic information processes of DNA replication, replication initiation, transcription, and translation which are solved stochastically and averaged over 1,000 replicates/cells. The model predicts the time required for replication initiation and DNA replication to be 8 and 50 min on average respectively and the number of proteins and ribosomal components to be approximately doubled in a cell cycle. The model of genetic information processing when combined with the essential metabolic and cell growth networks will provide a powerful platform for studying the fundamental principles of life., (Copyright © 2019 Thornburg, Melo, Bianchi, Brier, Crotty, Breuer, Smith, Hutchison, Glass and Luthey-Schulten.)

Novel site-specific attachment strategies combined with improvements of computational resources enable new insights into the mechanics of the monovalent biotin/streptavidin complex under load and forced us to rethink the diversity of rupture forces reported in the literature. We discovered that the mechanical stability of this complex depends strongly on the geometry in which force is applied. By atomic force microscopy-based single molecule force spectroscopy we found unbinding of biotin to occur beyond 400 pN at force loading rates of 10 nN/s when monovalent streptavidin was tethered at its C-terminus. This value is about twice as high than that for N-terminal attachment. Steered molecular dynamics simulations provided a detailed picture of the mechanics of the unbinding process in the corresponding force loading geometries. Using machine learning techniques, we connected findings from hundreds of simulations to the experimental results, identifying different force propagation pathways. Interestingly, we observed that depending on force loading geometry, partial unfolding of N-terminal region of monovalent streptavidin occurs before biotin is released from the binding pocket.

Hybrid methods that combine quantum mechanics (QM) and molecular mechanics (MM) can be applied to studies of reaction mechanisms in locations ranging from active sites of small enzymes to multiple sites in large bioenergetic complexes. By combining the widely used molecular dynamics and visualization programs NAMD and VMD with the quantum chemistry packages ORCA and MOPAC, we created an integrated, comprehensive, customizable, and easy-to-use suite (http://www.ks.uiuc.edu/Research/qmmm). Through the QwikMD interface, setup, execution, visualization, and analysis are streamlined for all levels of expertise.

Using protein counts sampled from single cell proteomics distributions to constrain fluxes through a genome-scale model of metabolism, Population flux balance analysis (Population FBA) successfully described metabolic heterogeneity in a population of independent Escherichia coli cells growing in a defined medium. We extend the methodology to account for correlations in protein expression arising from the co-regulation of genes and apply it to study the growth of independent Saccharomyces cerevisiae cells in two different growth media. We find the partitioning of flux between fermentation and respiration predicted by our model agrees with recent 13C fluxomics experiments, and that our model largely recovers the Crabtree effect (the experimentally known bias among certain yeast species toward fermentation with the production of ethanol even in the presence of oxygen), while FBA without proteomics constraints predicts respirative metabolism almost exclusively. The comparisons to the 13C study showed improvement upon inclusion of the correlations and motivated a technique to systematically identify inconsistent kinetic parameters in the literature. The minor secretion fluxes for glycerol and acetate are underestimated by our method, which indicate a need for further refinements to the metabolic model. For yeast cells grown in synthetic defined (SD) medium, the calculated broad distribution of growth rates matches experimental observations from single cell studies, and we characterize several metabolic phenotypes within our modeled populations that make use of diverse pathways. Fast growing yeast cells are predicted to perform significant amount of respiration, use serine-glycine cycle and produce ethanol in mitochondria as opposed to slow growing cells. We use a genetic algorithm to determine the proteomics constraints necessary to reproduce the growth rate distributions seen experimentally. We find that a core set of 51 constraints are essential but that additional constraints are still necessary to recover the observed growth rate distribution in SD medium.

Background: Molecular dynamics has emerged as an important research methodology covering systems to the level of millions of atoms. However, insufficient sampling often limits its application. The limitation is due to rough energy landscapes, with many local minima separated by high-energy barriers, which govern the biomolecular motion., Scope of Review: In the past few decades methods have been developed that address the sampling problem, such as replica-exchange molecular dynamics, metadynamics and simulated annealing. Here we present an overview over theses sampling methods in an attempt to shed light on which should be selected depending on the type of system property studied., Major Conclusions: Enhanced sampling methods have been employed for a broad range of biological systems and the choice of a suitable method is connected to biological and physical characteristics of the system, in particular system size. While metadynamics and replica-exchange molecular dynamics are the most adopted sampling methods to study biomolecular dynamics, simulated annealing is well suited to characterize very flexible systems. The use of annealing methods for a long time was restricted to simulation of small proteins; however, a variant of the method, generalized simulated annealing, can be employed at a relatively low computational cost to large macromolecular complexes., General Significance: Molecular dynamics trajectories frequently do not reach all relevant conformational substates, for example those connected with biological function, a problem that can be addressed by employing enhanced sampling algorithms. This article is part of a Special Issue entitled Recent developments of molecular dynamics., (Copyright © 2014 Elsevier B.V. All rights reserved.)