Machine Learning Approach to Vertical Energy Gap in Redox Processes.

A straightforward approach to calculating the free energy change (ΔG) and reorganization energy of a redox process is linear response approximation (LRA). However, accurate prediction of redox properties is still challenging due to difficulties in conformational sampling and vertical energy-gap sampling. Expensive hybrid quantum mechanical/molecular mechanical (QM/MM) calculations are typically employed in sampling energy gaps using conformations from simulations. To alleviate the computational cost associated with the expensive QM method in the QM/MM calculation, we propose machine learning (ML) methods to predict the vertical energy gaps (VEGs). We tested several ML models to predict the VEGs and observed that simple models like linear regression show excellent performance (mean absolute error ∼0.1 eV) in predicting VEGs in all test systems, even when using features extracted from cheaper semiempirical methods. Our best ML model (extra trees regressor) shows a mean absolute error of around 0.1 eV while using features from the cheapest QM method. We anticipate our approach can be generalized to larger macromolecular systems with more complex redox centers.

Variable Non-Gaussian Transport of Nanoplastic on Supported Lipid Bilayers in Saline Conditions.

Nanoplastic-lipid interaction is vital to understanding the nanoscale mechanism of plastic adsorption and aggregation on a lipid membrane surface. However, a single-particle mechanistic picture of the nanoplastic transport process on a lipid surface remains unclear. Here, we report a salt-dependent non-Gaussian transport mechanism of polystyrene particles on a supported 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer surface. Particle stickiness on the POPC surface increases with salt concentration, where the particles stay longer at the surface and diffuse to shorter distances. Additionally, a non-Gaussian diffusion state dominates the transport process at high salt concentrations. Our current study provides insight into the transport mechanism of polystyrene (PS) particles on supported lipid membranes, which is essential to understanding fundamental questions regarding the adsorption mechanisms of nanoplastics on lipid surfaces.

Many Roles of Carbohydrates: A Computational Spotlight on the Coronavirus S Protein Binding.

Glycosylation is one of the post-translational modifications with more than 50% of human proteins being glycosylated. The exact nature and chemical composition of glycans are inaccessible to X-ray or cryo-electron microscopy imaging techniques. Therefore, computational modeling studies and molecular dynamics must be used as a "computational microscope". The spike (S) protein of SARS-CoV-2 is heavily glycosylated, and a few glycans play a more functional role "beyond shielding". In this mini-review, we discuss computational investigations of the roles of specific S-protein and ACE2 glycans in the overall ACE2-S protein binding. We highlight different functions of specific glycans demonstrated in myriad computational models and simulations in the context of the SARS-CoV-2 virus binding to the receptor. We also discuss interactions between glycocalyx and the S protein, which may be utilized to design prophylactic polysaccharide-based therapeutics targeting the S protein. In addition, we underline the recent emergence of coronavirus variants and their impact on the S protein and its glycans.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

A General Strategy Toward pH-Resistant Phenolic Fluorophores for High-Fidelity Sensing and Bioimaging Applications.

Aryl alcohol-type or phenolic fluorophores offer diverse opportunities for developing bioimaging agents and fluorescence probes. Due to the inherently acidic hydroxyl functionality, phenolic fluorophores provide pH-dependent emission signals. Therefore, except for developing pH probes, the pH-dependent nature of phenolic fluorophores should be considered in bioimaging applications but has been neglected. Here we show that a simple structural remedy converts conventional phenolic fluorophores into pH-resistant derivatives, which also offer "medium-resistant" emission properties. The structural modification involves a single-step introduction of a hydrogen-bonding acceptor such as morpholine nearby the phenolic hydroxyl group, which also leads to emission bathochromic shift, increased Stokes shift, enhanced photo-stability and stronger emission for several dyes. The strategy greatly expands the current fluorophores' repertoire for reliable bioimaging applications, as demonstrated here with ratiometric imaging of cells and tissues.

Insights into substrate transport and water permeation in the mycobacterial transporter MmpL3.

Mycobacteria, such as Mycobacterium tuberculosis, are characterized by a uniquely thick and waxy cell envelope that consists of two membranes, with a variety of mycolates comprising their outer membrane (OM). The protein Mycobacterial membrane protein Large 3 (MmpL3) is responsible for the transport of a primary OM component, trehalose monomycolate (TMM), from the inner (cytoplasmic) membrane (IM) to the periplasmic space, a process driven by the proton gradient. Although multiple structures of MmpL3 with bound substrates have been solved, the exact pathway(s) for TMM or proton transport remains elusive. Here, employing molecular dynamics simulations we investigate putative pathways for either transport species. We hypothesized that MmpL3 will cycle through similar conformational states as the related transporter AcrB, which we used as targets for modeling the conformation of MmpL3. A continuous water pathway through the transmembrane region was found in one of these states, illustrating a putative pathway for protons. Additional equilibrium simulations revealed that TMM can diffuse from the membrane into a binding pocket in MmpL3 spontaneously. We also found that acetylation of TMM, which is required for transport, makes it more stable within MmpL3's periplasmic cavity compared with the unacetylated form.

SARS-CoV-2 spike opening dynamics and energetics reveal the individual roles of glycans and their collective impact.

The trimeric spike (S) glycoprotein, which protrudes from the SARS-CoV-2 viral envelope, binds to human ACE2, initiated by at least one protomer's receptor binding domain (RBD) switching from a "down" (closed) to an "up" (open) state. Here, we used large-scale molecular dynamics simulations and two-dimensional replica exchange umbrella sampling calculations with more than a thousand windows and an aggregate total of 160 µs of simulation to investigate this transition with and without glycans. We find that the glycosylated spike has a higher barrier to opening and also energetically favors the down state over the up state. Analysis of the S-protein opening pathway reveals that glycans at N165 and N122 interfere with hydrogen bonds between the RBD and the N-terminal domain in the up state, while glycans at N165 and N343 can stabilize both the down and up states. Finally, we estimate how epitope exposure for several known antibodies changes along the opening path. We find that the BD-368-2 antibody's epitope is continuously exposed, explaining its high efficacy.

Restoring and Enhancing the Potency of Existing Antibiotics against Drug-Resistant Gram-Negative Bacteria through the Development of Potent Small-Molecule Adjuvants.

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

Proton transfer activity of the reconstituted Mycobacterium tuberculosis MmpL3 is modulated by substrate mimics and inhibitors.

Transporters belonging to the Resistance-Nodulation-cell Division (RND) superfamily of proteins such as Mycobacterium tuberculosis MmpL3 and its analogs are the focus of intense investigations due to their importance in the physiology of Corynebacterium-Mycobacterium-Nocardia species and antimycobacterial drug discovery. These transporters deliver trehalose monomycolates, the precursors of major lipids of the outer membrane, to the periplasm by a proton motive force-dependent mechanism. In this study, we successfully purified, from native membranes, the full-length and the C-terminal truncated M. tuberculosis MmpL3 and Corynebacterium glutamicum CmpL1 proteins and reconstituted them into proteoliposomes. We also generated a series of substrate mimics and inhibitors specific to these transporters, analyzed their activities in the reconstituted proteoliposomes, and carried out molecular dynamics simulations of the model MmpL3 transporter at different pH. We found that all reconstituted proteins facilitate proton translocation across a phospholipid bilayer, but MmpL3 and CmpL1 differ dramatically in their responses to pH and interactions with substrate mimics and indole-2-carboxamide inhibitors. Our results further suggest that some inhibitors abolish the transport activity of MmpL3 and CmpL1 by inhibition of proton translocation.

A 300-fold conductivity increase in microbial cytochrome nanowires due to temperature-induced restructuring of hydrogen bonding networks.

Although proteins are considered as nonconductors that transfer electrons only up to 1 to 2 nanometers via tunneling, Geobacter sulfurreducens transports respiratory electrons over micrometers, to insoluble acceptors or syntrophic partner cells, via nanowires composed of polymerized cytochrome OmcS. However, the mechanism enabling this long-range conduction is unclear. Here, we demonstrate that individual nanowires exhibit theoretically predicted hopping conductance, at rate (>1010 s-1) comparable to synthetic molecular wires, with negligible carrier loss over micrometers. Unexpectedly, nanowires show a 300-fold increase in their intrinsic conductance upon cooling, which vanishes upon deuteration. Computations show that cooling causes a massive rearrangement of hydrogen bonding networks in nanowires. Cooling makes hemes more planar, as revealed by Raman spectroscopy and simulations, and lowers their reduction potential. We find that the protein surrounding the hemes acts as a temperature-sensitive switch that controls charge transport by sensing environmental perturbations. Rational engineering of heme environments could enable systematic tuning of extracellular respiration.

Resolving the Hydride Transfer Pathway in Oxidative Conversion of Proline to Pyrrole.

Thiotemplated pyrrole is a prevailing intermediate in the synthesis of numerous natural products in which the pyrrole is tethered to a carrier protein (CP). Biosynthesis of the pyrrole requires oxidation of an l-proline side chain. Herein, we investigate the biocatalytic mechanism of proline-to-pyrrole synthesis by molecular dynamics simulations, quantum mechanics/molecular mechanics simulations, and electronic structure calculations using the recently reported (Thapa, H. R., et al. Biochemistry 2019, 58, 918) structure of a type II nonribosomal protein synthetase (NRPS) Bmp3-Bmp1 (Oxidase-CP) complex. The substrate (l-proline) is attached to the Bmp1(CP), and the catalytic site is located inside the flavin-dependent oxidase (Bmp3). We show that the FAD isoalloxazine ring is stabilized in the catalytic site of Bmp3 by strong hydrogen bonding with Asn123, Ile125, Ser126, and Thr158. After the initial deprotonation followed by an enamine-imine tautomerization, oxidation of the C2-C3 or C2-N1 bond, through a hydride transfer (from either C3 or N1), is required for the pyrrole synthesis. Computational results indicate that the hydride transfer is more likely to occur from C3 than N1. Additionally, we demonstrate the elasticity in the oxidase active site through enzymatic synthesis of proline derivatives.

Inward-facing glycine residues create sharp turns in ß-barrel membrane proteins.

The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively ß-barrels composed of between 8 and 26 ß-strands. To explore the relationship between ß-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 ß-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the ß-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in ß-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in ß-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.

Machine Learning Reveals the Critical Interactions for SARS-CoV-2 Spike Protein Binding to ACE2.

SARS-CoV and SARS-CoV-2 bind to the human ACE2 receptor in practically identical conformations, although several residues of the receptor-binding domain (RBD) differ between them. Herein, we have used molecular dynamics (MD) simulations, machine learning (ML), and free-energy perturbation (FEP) calculations to elucidate the differences in binding by the two viruses. Although only subtle differences were observed from the initial MD simulations of the two RBD-ACE2 complexes, ML identified the individual residues with the most distinctive ACE2 interactions, many of which have been highlighted in previous experimental studies. FEP calculations quantified the corresponding differences in binding free energies to ACE2, and examination of MD trajectories provided structural explanations for these differences. Lastly, the energetics of emerging SARS-CoV-2 mutations were studied, showing that the affinity of the RBD for ACE2 is increased by N501Y and E484K mutations but is slightly decreased by K417N.

ACE2 glycans preferentially interact with SARS-CoV-2 over SARS-CoV.

We report a distinct difference in the interactions of the glycans of the host-cell receptor, ACE2, with SARS-CoV-2 and SARS-CoV S-protein receptor-binding domains (RBDs). Our analysis demonstrates that the ACE2 glycan at N322 enhances interactions with the SARS-CoV-2 RBD while the ACE2 glycan at N90 may offer protection against infections of both coronaviruses depending on its composition. The interactions of the ACE2 glycan at N322 with SARS-CoV RBD are blocked by the presence of the RBD glycan at N357 of the SARS-CoV RBD. The absence of this glycosylation site on SARS-CoV-2 RBD may enhance its binding with ACE2.

Gatekeeping Ketosynthases Dictate Initiation of Assembly Line Biosynthesis of Pyrrolic Polyketides.

Assembly line biosynthesis of polyketide natural products involves checkpoints where identities of thiotemplated intermediates are verified before polyketide extension reactions are allowed to proceed. Determining what these checkpoints are and how they operate is critical for reprogramming polyketide assembly lines. Here we demonstrate that ketosynthase (KS) domains can perform this gatekeeping role. By comparing the substrate specificities for polyketide synthases that extend pyrrolyl and halogenated pyrrolyl substrates, we find that KS domains that need to differentiate between these two substrates exercise high selectivity. We additionally find that amino acid residues in the KS active site facilitate this selectivity and that these residues are amenable to rational engineering. On the other hand, KS domains that do not need to make selectivity decisions in their native physiological context are substrate-promiscuous. We also provide evidence that delivery of substrates to polyketide synthases by non-native carrier proteins is accompanied by reduced biosynthetic efficiency.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease.

The main protease (M pro ) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of M pro , a cysteine protease, have been determined, facilitating structure-based drug design. M pro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, M pro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nu-cleophile Cys145 have been debated in previous studies of SARS-CoV M pro , but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 M pro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of M pro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α -ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 M pro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

Electric field stimulates production of highly conductive microbial OmcZ nanowires.

Multifunctional living materials are attractive due to their powerful ability to self-repair and replicate. However, most natural materials lack electronic functionality. Here we show that an electric field, applied to electricity-producing Geobacter sulfurreducens biofilms, stimulates production of cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S cm-1) and threefold higher stiffness (1.5 GPa) than the cytochrome OmcS nanowires that are important in natural environments. Using chemical imaging-based multimodal nanospectroscopy, we correlate protein structure with function and observe pH-induced conformational switching to ß-sheets in individual nanowires, which increases their stiffness and conductivity by 100-fold due to enhanced π-stacking of heme groups; this was further confirmed by computational modeling and bulk spectroscopic studies. These nanowires can transduce mechanical and chemical stimuli into electrical signals to perform sensing, synthesis and energy production. These findings of biologically produced, highly conductive protein nanowires may help to guide the development of seamless, bidirectional interfaces between biological and electronic systems.

The Effect of (-)-Epigallocatechin-3-Gallate on the Amyloid-ß Secondary Structure.

Amyloid-ß (Aß) is a macromolecular structure of great interest because its misfolding and aggregation, along with changes in the secondary structure, have been correlated with its toxicity in various neurodegenerative diseases. Small drug-like molecules can modulate the amyloid secondary structure and therefore have raised significant interest in applications to active and passive therapies targeting amyloids. In this study, we investigate the interactions of epigallocatechin-3-gallate (EGCG), found in green tea, with Aß polypeptides, using a combination of in vitro immuno-infrared sensor measurements, docking, molecular dynamics simulations, and ab initio calculations. We find that the interactions of EGCG are dominated by only a few residues in the fibrils, including hydrophobic π-π interactions with aromatic rings of side chains and hydrophilic interactions with the backbone of Aß, as confirmed by extended (1-µs-long) molecular dynamics simulations. Immuno-infrared sensor data are consistent with degradation of Aß fibril induced by EGCG and inhibition of Aß fibril and oligomer formation, as manifested by the recovery of the amide-I band of monomeric Aß, which is red-shifted by 26 cm-1 when compared to the amide-I band of the fibrillar form. The shift is rationalized by computations of the infrared spectra of Aß42 model structures, suggesting that the conformational change involves interchain hydrogen bonds in the amyloid fibrils that are broken upon binding of EGCG.

Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics.

CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.