Correction for 'Mechanism of RGD-conjugated nanodevice binding to its target protein integrin αVβ3 by atomistic molecular dynamics and machine learning' by Giulia Frigerio et al., Nanoscale, 2024, 16, 4063-4081, https://doi.org/10.1039/D3NR05123D.

[FeFe]-hydrogenases are highly efficient enzymes in the reversible catalysis of molecular hydrogen production and oxidation. Their active site, the H-cluster, consists of a [4Fe-4S]H subcluster linked to a binuclear [2Fe]H organometallic unit. Many [FeFe]-hydrogenases, such as the one from Desulfovibrio desulfuricans (DdHydAB), possess accessory Fe-S clusters (F and F') that mediate electron transfer. This study employs hybrid quantum mechanics/molecular mechanics (QM/MM) methods to characterize the electronic structure and thermodynamic landscape associated with redox and protonation events in the complete Fe-S cluster network of DdHydAB. Our calculations indicate that the F' cluster plays a key role in the initial reduction of the oxidized resting state, acting as the preferential site for the accumulation of the first electron. Analysis of protonated states, upon reduction events, reveals a strong correlation between protonation and electron transfer (PCET), with protonation at the H-cluster inducing electron transfer from the F' cluster to the H-cluster. Calculations indicate that the formation of a terminal hydride is energetically favored over ADT protonation, and subsequent isomerization to a bridging hydride (μ-H) is further stabilizing, albeit potentially kinetically limiting. The study highlights how accessory clusters influence the electronic distribution and redox properties of the H-cluster, underscoring the importance of considering the entire Fe-S cluster system for a complete understanding of the catalytic mechanism of [FeFe]-hydrogenases.

The art of designing coupling systems to drive reactions for endergonic synthesis is a subject of great interest in the scientific community, but it still presents major challenges. The aim of this kinetic study was to run simulations in COPASI 4.39 to test the behavior of hypothetical models for a system that couples two independent reactions, one exergonic and the other endergonic. In our computational study, we unraveled the qualitative and quantitative conditions that allow and benefit coupling, considering all possible reaction pathways within the network. Optimal conditions were reached by assigning favorable directionalities and low activation energies to six reaction steps within a network that featured twenty reaction steps. Moreover, different models were designed and tested in order to investigate the availability of coupling with different reaction steps.

Mycoplasma pneumoniae (Mpn, class Mollicutes) is both an important human pathogen and a model organism. We performed a proteome-wide investigation of intrinsically disordered regions (IDRs) in Mpn. Compared to other bacteria, a considerable fraction of the Mpn proteome (17%) is embedded in IDRs, which are abundant in membrane, non-essential proteins, as well as in proteins that mediate cytoadherence and virulence. Notably, proteins that form the attachment organelle, a specialized structure, are particularly rich in IDRs. Likewise, analysis of protein architectures indicated that some Mollicute-specific domains are preferentially associated with IDRs. Perusal of proteome-wide data also revealed that, as in eukaryotes, structural disorder associates with higher protein degradation rates and that Mpn IDRs are preferential targets of phosphorylation. When we investigated the ensemble features for Mpn IDRs, we used two predictors and benchmarked the results using coarse-grained simulations. We found that ensemble properties are mediated by similar sequence features as in eukaryotes, so that compact IDRs tend to have high residue stickiness, high hydropathy decoration, and few charged residues. We also found that IDRs in attachment organelle proteins are particularly extended and display high conformational entropy. We suggest that these features are exploited for motility through the generation of an entropic force. In summary, our results suggest that structural disorder contributes to very specialized functions in Mpn. Our data also highlight the functional relevance of IDRs, as the minimal proteome of this model organism displays a considerable level of structural disorder.IMPORTANCEWe performed a proteome-wide investigation of intrinsically disordered regions (IDRs) in Mycoplasma pneumoniae (Mpn, class Mollicutes). A considerable fraction of the Mpn proteome (17%) is embedded in IDRs, which tend to be associated with Mollicute-specific domains and are abundant in membrane, non-essential proteins, as well as in proteins that mediate cytoadherence and virulence. As in eukaryotes, structural disorder associates with higher protein degradation rates, and Mpn IDRs are preferential targets of phosphorylation. The ensemble properties of Mpn IDRs are mediated by similar sequence features as in eukaryotes, and IDRs in attachment organelle proteins display high conformational entropy. We suggest that this feature is exploited for motility through the generation of an entropic force. In summary, we show that structural disorder contributes to very specialized functions in Mpn. Our data highlight the functional relevance of IDRs, as the minimal proteome of this model organism displays a considerable level of structural disorder.

Iron-Sulfur (Fe-S) proteins play essential roles in a wide range of biological processes, from energy conversion and respiration to DNA repair and redox signaling, making them highly relevant to both bioenergetics and human health. These proteins mediate electron transfer through finely tuned reduction potentials (RP) defined by their metal cofactors. However, predicting RP from protein structures remains a significant challenge due to the complex electronic nature of Fe-S clusters and their intricate coupling with the surrounding protein environment. This complexity limits our ability to systematically modulate RP, hindering efforts in high-throughput and rational protein design. In this study, we introduce a Machine Learning (ML) framework, FeS-RedPred, for accurate and scalable prediction of RP in Fe-S proteins. We focus on mono- and binuclear clusters, such as rubredoxins and [2Fe-2S] clusters of ferredoxins, Rieske, and mitoNEET-type, which serve as ideal model systems thanks to the availability of abundant structural and electrochemical data. Our approach relies on structure-derived molecular descriptors computed across multiple spatial scales, from local atomic environments to global protein-level features. Using Extreme Gradient Boosting (XGB) models, we achieve a mean absolute error of ∼40 mV, which is competitive with state-of-the-art computational approaches, while also providing a highly efficient compromise between accuracy and computational cost. Beyond predictive accuracy, our model also offers indications about the determinants of RP, enabling a basis for interpretation and potentially guiding protein engineering. This work provides a valuable foundation for understanding the redox behavior of metalloproteins, enabling the high-throughput prediction of redox potentials and informing data-driven design across diverse protein families.

BACKGROUND:Intrinsically disordered protein regions (IDRs) are implicated in diverse cellular processes in eukaryotes and, in these organisms, they cover up to 40% of the proteome. Surprisingly little is known about IDRs in bacterial proteomes. Specifically, a number of questions remain unanswered, such as the role of these regions in host-pathogen interactions, their adaptive potential and evolutionary trajectories, as well as their biophysical properties. Here we focus on Mycobacterium tuberculosis and take advantage of the fact that, due to its extreme epidemiological relevance, several large-scale analyses are available. RESULTS:After benchmarking different disorder prediction tools, we integrate multiple levels of biological information to show that IDR-containing proteins are involved in virulence, in the modulation of host immune response, and in lipid metabolism. Mycobacterium tuberculosis IDRs are fast evolving and poorly antigenic, and they display specific sequence-ensemble-function relationships. Conversely, human proteins that interact with Mycobacterium tuberculosis are evolutionary constrained, widely expressed, and highly connected in the human interactome map. This indicates that the classical arms race paradigm is not universal in host-pathogen interactions. We also extend analysis to 540 human-infecting bacteria and we underscore wide variations in IDR representation and conformational properties. CONCLUSIONS:Our data point to a role of IDRs in contributing to bacterial virulence, interaction with the human host, and control of immune responses. Although this awaits experimental validation, we suggest that Mycobacterium tuberculosis also uses IDRs to sense and interact with its environment. Herein, we provide a database of bacterial IDRs, together with relevant parameters, for public use.

The performance of artificial molecular machines relies on the interplay between molecular design and environmental factors, yet how solvation shapes their energy landscapes and kinetics remains poorly understood. Here, we combine well-tempered and infrequent metadynamics to investigate equilibrium shuttling in a minimal [2]rotaxane inspired by Borsley's fuel-driven molecular motor. By systematically varying solvent polarity and hydrogen-bonding capacity, we uncover distinct thermodynamic and kinetic regimes that govern macrocycle motion. In highly polar, hydrogen-bond-accepting media, the macrocycle adopts a symmetric distribution between binding sites, with enthalpic and entropic forces in direct competition. Conversely, in low-polarity, hydrogen-bond-donating environments, the axle undergoes a conformational collapse that entropically biases occupancy toward a single station in the absence of chemical fuel. Despite comparable free-energy barriers across conditions (9-13 kcal/mol), the transition pathways exhibit pronounced solvent-dependent asymmetries and energetic ruggedness. These findings provide a molecular-level framework for understanding how solvation dictates passive ratchet behavior and offer strategic insights for designing high-performance molecular machines tailored to complex media.

Laccases that oxidize low-density polyethylene (LDPE) represent a promising strategy for bioremediation purposes. To rationalize or optimize their PE-oxidative activity, two fundamental factors must be considered: the enzyme‘s redox potential and its binding affinity/mode towards LDPE. Indeed, a stable laccase-PE complex may facilitate a thermodynamically unfavorable electron transfer, even without redox mediators. In this study, we compared the redox potential and the LDPE-binding properties of three different PE-oxidizing laccases: a fungal high-redox potential laccase from Trametes versicolor , a bacterial low-redox potential laccase from Bacillus subtilis , and the recently characterized LMCO2 from Rhodococcus opacus R7. First we found that LMCO2 is a low-potential laccase (E°=413 mV), as reported in other bacterial variants. Using computational tools, we simulated the interactions of these laccases with a large LDPE model and highlighted the key role of hydrophobic residues surrounding the T1 site. Notably, a methionine-rich loop in LMCO2 appears to enhance the formation of a stable complex with LDPE, potentially facilitating electron transfer. This study underscores the necessity for comprehensive computational strategies to analyze enzyme-polymer interactions beyond simplistic models, uncovering critical binding determinants and informing future mutagenesis experiments, in order to enhance laccase performance and rationalize variations in enzymatic activity.

The origin of recently reported anomalous fluorescence emissions from aqueous solutions of nonaromatic solutes remains elusive. To determine whether the solute nature influences the fluorescence characteristics and to identify a potential common mechanism, we measured the fluorescence spectra of 21 different solutions. We observed similar emission characteristics across all samples, suggesting that the solute nature plays a minimal role in the emission mechanism. Using time-dependent density functional theory on large water, NaCl/water, and glycerol/water clusters, we attributed the anomalous emission to the decay of charge-transfer-to-solvent excitations (CTTS) which populate a diradical zwitterionic excited state localized at hydrogen-bond network defects. The Arrhenius-like plots for NaCl and glycerol solutions revealed that the S1 nonradiative decay pathway involves the diradical recombination via librational motion. We propose that the presence of solute molecules slows this process, thus increasing the lifetime of the CTTS excited states and facilitating emission.

Several mammalian genes have originated from the domestication of retrotransposons, selfish mobile elements related to retroviruses. Some of the proteins encoded by these genes have maintained virus-like features; including self-processing, capsid structure formation, and the generation of different isoforms through -1 programmed ribosomal frameshifting. Using quantitative approaches in molecular evolution and biophysical analyses, we studied 28 retrotransposon-derived genes, with a focus on the evolution of virus-like features. By analyzing the rate of synonymous substitutions, we show that the -1 programmed ribosomal frameshifting mechanism in three of these genes (PEG10, PNMA3, and PNMA5) is conserved across mammals and originates alternative proteins. These genes were targets of positive selection in primates, and one of the positively selected sites affects a B-cell epitope on the spike domain of the PNMA5 capsid, a finding reminiscent of observations in infectious viruses. More generally, we found that retrotransposon-derived proteins vary in their intrinsically disordered region content and this is directly associated with their evolutionary rates. Most positively selected sites in these proteins are located in intrinsically disordered regions and some of them impact protein posttranslational modifications, such as autocleavage and phosphorylation. Detailed analyses of the biophysical properties of intrinsically disordered regions showed that positive selection preferentially targeted regions with lower conformational entropy. Furthermore, positive selection introduces variation in binary sequence patterns across orthologues, as well as in chain compaction. Our results shed light on the evolutionary trajectories of a unique class of mammalian genes and suggest a novel approach to study how intrinsically disordered region biophysical characteristics are affected by evolution.

Active targeting strategies have been proposed to enhance the selective uptake of nanoparticles (NPs) by diseased cells, and recent experimental findings have proven the effectiveness of this approach. However, no mechanistic studies have yet revealed the atomistic details of the interactions between ligand-activated NPs and integrins. As a case study, here we investigate, by means of advanced molecular dynamics simulations (MD) and machine learning methods (namely equilibrium MD, binding free energy calculations and training of self-organized maps), the interaction of a cyclic-RGD-conjugated PEGylated TiO2 NP (the nanodevice) with the extracellular segment of integrin αVβ3 (the target), the latter experimentally well-known to be over-expressed in several solid tumors. Firstly, we proved that the cyclic-RGD ligand binding to the integrin pocket is established and kept stable even in the presence of the cumbersome realistic model of the nanodevice. In this respect, the unsupervised machine learning analysis allowed a detailed comparison of the ligand/integrin binding in the presence and in the absence of the nanodevice, which unveiled differences in the chemical features. Then, we discovered that unbound cyclic RGDs conjugated to the NP largely contribute to the interactions between the nanodevice and the integrin. Finally, by increasing the density of cyclic RGDs on the PEGylated TiO2 NP, we observed a proportional enhancement of the nanodevice/target binding. All these findings can be exploited to achieve an improved targeting selectivity and cellular uptake, and thus a more successful clinical outcome.

Laccases (EC 1.10.3.2) are multicopper oxidases with the capability to oxidize diverse phenolic and non-phenolic substrates. While the molecular mechanism of their activity towards phenolic substrates is well-established, their reactivity towards non-phenolic substrates, such as polycyclic aromatic hydrocarbons (PAHs), remains unclear. To elucidate the oxidation mechanism of PAHs, particularly the activation mechanism of the sp(2) aromatic C-H bond, we conducted a density functional theory investigation on the oxidation of two PAHs (anthracene and benzo[a]pyrene) using an extensive model of the T1 copper catalytic site of the fungal laccase from Trametes versicolor.

The design of protein-metal complexes is rapidly advancing, with applications spanning catalysis, sensing, and bioremediation. We report a comprehensive investigation of METPsc1, a Miniaturized Electron Transfer Protein, in complex with cadmium. This study elucidates the impact of metal coordination on protein folding and structural dynamics across temperatures from 100 K to 300 K. Our findings reveal that METPsc1, composed of two similar halves stabilized by intramolecular hydrogen bonds, exhibits a unique "clothespin-like" recoil mechanism. This allows it to adapt to metal ions of varying radii, mirroring the flexibility observed in natural rubredoxins. High-resolution crystallography and molecular dynamics simulations unveil concerted backbone motions and subtle temperature-dependent shifts in side-chain conformations, particularly for residues involved in crystal packing. Notably, CdS bond lengths increase with temperature, correlating with anisotropic motions of the sulfur atoms involved in second-shell hydrogen bonding. This suggests a dynamic role of protein matrix upon redox cycling. These insights into METPsc1 highlight its potential for catalysis and contribute to the designing of artificial metalloproteins with functional plasticity.

Three hexacarbonyl diiron dithiolate complexes [Fe-2(CO)(6)(mu-(SCH2)(2)X)] with different substituted bridgeheads (X=CH2, CEt2, CBn2 (Bn=CH2C6H5)), have been studied under the same experimental conditions by cyclic voltammetry in dichloromethane [NBu4][PF6] 0.2 M. DFT calculations were performed to rationalize the mechanism of reduction of these compounds. The three complexes undergo a two-electron transfer whose the mechanism depends on the bulkiness of the dithiolate bridge, which involves a different timing of the structural changes (Fe-S bond cleavage, inversion of conformation and CO bridging) vs redox steps. The introduction of a bulky group in the dithiolate linker has obviously an effect on normally ordered (as for propanedithiolate (pdt)) or inverted (pdt(Et2), pdt(Bn2)) reduction potentials. Et -> Bn replacement is not theoretically predicted to alter the geometry and energy of the most stable mono-reduced and bi-reduced forms but such a replacement alters the kinetics of the electron transfer vs the structural changes.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

In endemic regions (West Africa and the Congo Basin), the genetic diversity of monkeypox virus (MPXV) is geographically structured into two major clades (Clades I and II) that differ in virulence and host associations. Clade IIb is closely related to the B.1 lineage, which is dominating a worldwide outbreak initiated in 2022. Lineage B.1 has however accumulated mutations of unknown significance that most likely result from apolipoprotein B mRNA editing catalytic polypeptide-like 3 (APOBEC3) editing. We applied a population genetics-phylogenetics approach to investigate the evolution of MPXV during historical viral spread in Africa and to infer the distribution of fitness effects. We observed a high preponderance of codons evolving under strong purifying selection, particularly in viral genes involved in morphogenesis and replication or transcription. However, signals of positive selection were also detected and were enriched in genes involved in immunomodulation and/or virulence. In particular, several genes showing evidence of positive selection were found to hijack different steps of the cellular pathway that senses cytosolic DNA. Also, a few selected sites in genes that are not directly involved in immunomodulation are suggestive of antibody escape or other immune-mediated pressures. Because orthopoxvirus host range is primarily determined by the interaction with the host immune system, we suggest that the positive selection signals represent signatures of host adaptation and contribute to the different virulence of Clade I and II MPXVs. We also used the calculated selection coefficients to infer the effects of mutations that define the predominant human MPXV1 (hMPXV1) lineage B.1, as well as the changes that have been accumulating during the worldwide outbreak. Results indicated that a proportion of deleterious mutations were purged from the predominant outbreak lineage, whose spread was not driven by the presence of beneficial changes. Polymorphic mutations with a predicted beneficial effect on fitness are few and have a low frequency. It remains to be determined whether they have any significance for ongoing virus evolution.

Oxidative stress and metal dyshomeostasis are considered crucial factors in the pathogenesis of Alzheimer’s disease (AD). Indeed, transition metal ions such as Cu(II) can generate reactive oxygen species (ROS) via O2 Fenton-like reduction, catalyzed by Cu(II) coordinated to the amyloid-beta (Aβ) peptide. Despite intensive efforts, the mechanisms of ROS-induced molecular damage remain poorly understood. In the present paper, we investigate, on the basis of Density Functional Theory (DFT) computations, a possible mechanism of the OH radical propagation toward membrane phospholipid polar head and fatty acid chains starting from the end-product of the OH radical generation by Cu(II)-Aβ. Using phosphatidylcholine as a model of a single unit inside a membrane, we evaluated the thermochemistry of the OH propagation with the oxidation of a C-H bond and the formation of the radical moiety. The DFT results show that Cu(II)-Aβ-OH can oxidize only sn-2 C-H bonds of the polar head and can easily oxidize the C-H bond adjacent to the carbon–carbon double bond in a mono or bis unsaturated fatty acid chain. These results are discussed on the basis of the recent literature on in vitro Aβ metal-catalyzed oxidation and on the possible implications in the AD oxidative stress mechanism.

Nitrogen gas is a highly inert molecule and its activation under mild conditions represents a crucial goal in current research. In a recent study, the discovery of low-valence Ca(I) compounds capable of coordinating and reducing N2 was reported [B. Rösch, T. X. Gentner, J. Langer, C. Färber, J. Eyselein, L. Zhao, C. Ding, G. Frenking and S. Harder, Science, 2021, 371, 1125]. The study of low-valence alkaline earth complexes represents a new horizon in inorganic chemistry and demonstrates examples of spectacular reactivity. For example, complexes of the [BDI]2Mg2 type are selective reducing reagents in both organic and inorganic synthesis reactions. To date, however, no activity of Mg(I) complexes in the activation of the nitrogen molecule has been reported. By computational studies, in the present work, we investigated the analogies and differences of low-valence Ca(I) and Mg(I) complexes in the coordination, activation and protonation of N2. We have shown that the possibility of alkaline earth metals to employ atomic orbitals of the d type is reflected in the differences in the N2 binding energy and its coordination mode (end-on vs. side-on), as well as in the spin state of the resulting adduct (singlet vs. triplet). These divergences are finally observed in the subsequent protonation reaction, which turned out to be prohibitive in the presence of Mg.

Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.

Two complexes, related to the active site of [FeFe]-hydrogenases, [Fe2(CO)4(κ2-pma)(µ-bdt)] (1) and [Fe2(CO)4(κ2-pma)(µ-pdt)] (2) (bdt = benzene-1,2-dithiolate, pdt = propane-1,2-dithiolate) featuring the diaza chelate ligand trans-N-(2-pyridylmethylene)aniline (pma) were prepared, in order to study the influence of such a redox ligand, potentially non-innocent, on their redox behaviours. Both complexes were synthesized by photolysis in moderate yields, and they were characterized by IR, 1H and 13C{1H} NMR spectroscopies, elemental analyses and X-ray diffraction. Their electrochemical study by cyclic voltammetry, in the presence and in the absence of protons, revealed different behaviours depending on the aliphatic or aromatic nature of the dithiolate bridge. Density functional theory (DFT) calculations showed the role of the pma ligand as an electron reservoir, allowing the rationalization of the proton reduction process of complex 1.