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Oligogalacturonides (OGs) are pectin fragments released from the breakdown of the homogalacturonan during pathogenesis that act as Damage-Associated Molecular Patterns. OG-oxidase 1 (OGOX1) is an Arabidopsis berberine bridge enzyme-like (BBE-l) oligosaccharide oxidase that oxidizes OGs, impairing their elicitor activity and concomitantly releasing H

Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today’s oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S4 state—which was postulated half a century ago1 and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S4 state as the oxygen-radical state; its formation is followed by fast O–O bonding and O2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems.

Water oxidation occurring in the first steps of natural oxygenic photosynthesis is catalyzed by the pigment/protein complex Photosystem II. This process takes place on the Mn4Ca cluster located in the core of Photosystem II and proceeds along the five steps (S0-S4) of the so-called Kok-Joliot cycle until the release of molecular oxygen. The catalytic cycle can therefore be started afresh through insertion of a new water molecule. Here, combining quantum mechanics/molecular mechanics simulations and minimum energy path calculations, we characterized on different spin surfaces the events occurring in the last sector of the catalytic cycle from structural, electronic, and thermodynamic points of view. We found that the process of oxygen evolution and water insertion can be described well by a two-step mechanism, with oxygen release being the rate-limiting step of the process. Moreover, our results allow us to identify the upcoming water molecule required to regenerate the initial structure of the Mn4Ca cluster in the S0 state. The insertion of the water molecule was found to be coupled with the transfer of a proton to a neighboring hydroxide ion, thus resulting in the reconstitution of the most widely accepted model of the S0 state.

In the first steps of natural oxygenic photosynthesis, Sun light is used to oxidize water molecules into protons, electrons and molecular oxygen. This reaction takes place on the Mn$_4$Ca cluster located in the reaction centre of Photosystem II (PSII), where the cluster is assembled and continuously repaired through a process known as photoactivation. Understanding the molecular details of such process has important implications in different fields, in particular inspiring synthesis and repair strategies for artificial photosynthesis devices. In this regard, a detailed structural and dynamic characterization of the Photosystem II lacking Mn$_4$Ca cluster, namely $apo$ PSII, is a prerequisite for the full comprehension of the photoactivation. Recently, the structure of the $apo$ PSII was resolved at 2.55~\AA~resolution [Zhang \textit{et al., eLIFE}, 2017, \textbf{6}, e26933], suggesting a pre-organized structure of the protein cavity hosting the cluster. Anyway, the question whether these findings are a feature of the method used remains open. Here, by means of classical Molecular Dynamics simulations, we characterized structural and dynamic features of the $apo$ PSII for different protonation states of the cluster cavity. Albeit an overall conformational stability common to all investigated systems, we found significant deviations in the conformation of the side chains of the active site with respect to the x-ray positions. Our findings suggest that not all residues acting as Mn ligands are pre-organized prior the Mn$_4$Ca formation and previous local conformational changes are required in order to bind the first Mn ion in the high-affinity binding site.

The σ1 receptor (σ1-R) is an enigmatic endoplasmic reticulum resident transmembrane protein implicated in a variety of central nervous system disorders and whose agonists have neuroprotective activity. In spite of σ1-R’s physio-pathological and pharmacological importance, two of the most important features required to fully understand σ1-R function, namely the receptor endogenous ligand(s) and the molecular mechanism of ligand access to the binding site, have not yet been unequivocally determined. In this work, we performed molecular dynamics (MD) simulations to help clarify the potential route of access of ligand(s) to the σ1-R binding site, on which discordant results had been reported in the literature. Further, we combined computational and experimental procedures (i.e., virtual screening (VS), electron density map fitting and fluorescence titration experiments) to provide indications about the nature of σ1-R endogenous ligand(s). Our MD simulations on human σ1-R suggested that ligands access the binding site through a cavity that opens on the protein surface in contact with the membrane, in agreement with previous experimental studies on σ1-R from Xenopus laevis. Additionally, steroids were found to be among the preferred σ1-R ligands predicted by VS, and 16,17-didehydroprogesterone was shown by fluorescence titration to bind human σ1-R, with significantly higher affinity than the prototypic σ1-R ligand pridopidine in the same essay. These results support the hypothesis that steroids are among the most important physiological σ1-R ligands.

The COVID-19 pandemic continues to have a threatening impact on a global scale, largely due to the emergence of newly SARS-CoV-2 variants. The Mu (PANGO lineage B.1.621), was first identified in Colombia in January 2021 and was classified as a variant of interest (VOI) in August 2021, due to a constellation of mutations that likely-mediate an unexpectedly enhanced immune resistance to inactivated vaccine-elicited antibodies. Despite recent studies suggesting that the Mu variant appears to have less infectivity than the Delta variant, here we examined the structural effect of the Mu spike protein mutations and predicted the potential impact on infectivity of the Mu variant compared with the Delta and Delta plus spike protein.

In the first steps of natural oxygenic photosynthesis, sunlight is used to oxidize water molecules to protons, electrons and molecular oxygen. This reaction takes place on the Mn

Background Huntington’s Disease (HD) is a devastating and presently untreatable neurodegenerative disease, characterized by progressively disabling motor and mental manifestations. Sigma-1 receptor (σ1R), whose 3D structure has been recently determined by X-ray crystallography, is expressed in the central nervous system, and σ1R agonists have been shown to possess neuroprotective activity in neurodegenerative diseases. Aims Our overall aim is to exploit σ1R neuroprotective activity for HD therapy. Methods We are integrating computational and experimental methods: i) Virtual Screening (VS) of compound libraries available through the ZINC database towards σ1R; ii) Experimental ligand binding to purified σ1R in vitro by Surface Plasmon Resonance; iii) Assessment of ligand ability to improve the growth of fibroblasts obtained from HD patients, which is significantly impaired with respect to control cells. Additionally, we are performing Molecular Dynamics (MD) simulations to elucidate the mechanism of ligand entrance to σ1R binding site. Results i) Six known drugs have been demonstrated to be able to bind purified σ1R in vitro and improve survival and growth of HD fibroblasts; ii) Several human metabolites have been predicted by VS to bind σ1R. iii) Predictions on routes of ligand entrance have been provided by MD studies. Conclusions Our results support the validity of σ1R as a molecular target for HD therapy, and of the drug repositioning procedure implemented herein for the identification of new therapeutic agents against HD. Experimental validation of metabolites selected by VS and results of MD simulations will contribute to identify endogenous σ1R ligand(s) and mechanism of σ1R entrance.

Water oxidation occurring in the first steps of natural oxygenic photosynthesis is catalyzed by the pigment/protein complex Photosystem II. This process takes place on the Mn

Conversion of adenosine triphosphate (ATP) to the second messenger cyclic adenosine monophosphate (cAMP) is an essential reaction mechanism that takes place in eukaryotes, triggering a variety of signal transduction pathways. ATP conversion is catalyzed by the enzyme adenylyl cyclase (AC), which can be regulated by binding inhibitory, Gαi, and stimulatory, Gαs subunits. In the past twenty years, several crystal structures of AC in isolated form and complexed to Gαs subunits have been resolved. Nevertheless, the molecular basis of the inhibition mechanism of AC, induced by Gαi, is still far from being fully understood. Here, classical molecular dynamics simulations of the isolated holo AC protein type 5 and the holo binary complex AC5:Gαi have been analyzed to investigate the conformational impact of Gαi association on ATP-bound AC5. The results show that Gαi appears to inhibit the activity of AC5 by preventing the formation of a reactive ATP conformation.

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1.

GabR is a bacterial transcription regulator with a dimeric structure in which each subunit includes a wHTH (winged Helix-Turn-Helix) domain connected through a peptide linker to a large C-terminal domain folded as the enzyme aspartate aminotransferase (AAT). In Bacillus subtilis, GabR activates the genes involved in the metabolism of γ-amino butyric acid (GABA) upon formation of a PLP-GABA adduct. Recently, the crystallographic structure of an asymmetric form of GabR has been solved. This form (semi-holo) has one active site binding PLP as internal aldimine and the other the PLP-GABA complex. This work reports a molecular dynamics (MD) study aimed at understanding the characteristics of the asymmetric GabR form and compare them to the dynamics properties of previously studied symmetric holo (internal PLP aldimine at both active sites) and holo-GABA (containing PLP-GABA adducts) GabRs. Standard molecular dynamics and data analysis techniques have been used. The results indicate a remarkable asymmetry in the mobility and interactions of the different structural portions of the semi-holo GabR and of a few residues at the active site. The pattern is different from that observed in the other symmetrical GabR forms. The asymmetric perturbation of the active site residues may suggest the existence of a form of allosteric interference between the two active sites.

Water oxidation occurring in oxygenic natural photosynthesis is catalyzed by the protein-cofactor complex Photosystem II. The catalytic cycle leading to the water splitting into protons, electrons and molecular oxygen, consists of five consecutive steps, namely S0-S4. Here, using QM/MM Molecular Dynamics simulations, we characterized structural and magnetic properties of the putative S4 state prior to O2 release, considering two different spin multiplicities. Our results show an overall stability for both simulated systems in the ps timescale. Nonetheless, analysis of structural and magnetic properties identified discrepancies between the two simulations, conceivably having different consequences on the O2 evolution.

Long-term potentiation and depression of synaptic activity in response to stimuli is a key factor in reinforcement learning. Strengthening of the corticostriatal synapses depends on the second messenger cAMP, whose synthesis is catalysed by the enzyme adenylyl cyclase 5 (AC5), which is itself regulated by the stimulatory G alpha(olf) and inhibitory G alpha(i) proteins. AC isoforms have been suggested to act as coincidence detectors, promoting cellular responses only when convergent regulatory signals occur close in time. However, the mechanism for this is currently unclear, and seems to lie in their diverse regulation patterns. Despite attempts to isolate the ternary complex, it is not known if G alpha(olf) and G alpha(i) can bind to AC5 simultaneously, nor what activity the complex would have. Using protein structure-based molecular dynamics simulations, we show that this complex is stable and inactive. These simulations, along with Brownian dynamics simulations to estimate protein association rates constants, constrain a kinetic model that shows that the presence of this ternary inactive complex is crucial for AC5's ability to detect coincident signals, producing a synergistic increase in cAMP. These results reveal some of the prerequisites for corticostriatal synaptic plasticity, and explain recent experimental data on cAMP concentrations following receptor activation. Moreover, they provide insights into the regulatory mechanisms that control signal processing by different AC isoforms., Author summary Adenylyl cyclases (ACs) are enzymes that can translate extracellular signals into the intracellular molecule cAMP, which is thus a 2nd messenger of extracellular events. The brain expresses nine membrane-bound AC variants, and AC5 is the dominant form in the striatum. The striatum is the input stage of the basal ganglia, a brain structure involved in reward learning, i.e. the learning of behaviors that lead to rewarding stimuli (such as food, water, sugar, etc). During reward learning, cAMP production is crucial for strengthening the synapses from cortical neurons onto the striatal principal neurons, and its formation is dependent on several neuromodulatory systems such as dopamine and acetylcholine. It is, however, not understood how AC5 is activated by transient (subsecond) changes in the neuromodulatory signals. Here we combine several computational tools, from molecular dynamics and Brownian dynamics simulations to bioinformatics approaches, to inform and constrain a kinetic model of the AC5-dependent signaling system. We use this model to show how the specific molecular properties of AC5 can detect particular combinations of co-occuring transient changes in the neuromodulatory signals which thus result in a supralinear/synergistic cAMP production. Our results also provide insights into the computational capabilities of the different AC isoforms.

Adenylyl cyclase (AC) generates cyclic AMP required for a variety of cellular functions, and its regulation plays a major role in cellular signal transduction in eukaryotes and prokaryotes. All membrane-bound AC isoforms in eukaryotes can be activated by stimulatory G-proteins, but only AC1, AC5, and AC6 can be both stimulated and inhibited by active G alpha subunits, G alpha(s) and G(alpha)i, respectively. In principle, these G alpha(r) sensitive AC isoforms could form both binary and ternary complexes with G alpha subunits due to the noncompetitive association of inhibitory and stimulatory G alpha. However, the formation and possible catalytic activity of a putative ternary complex have not yet been experimentally confirmed due to its proposed short-lived nature. Here, the catalytic activity of such a ternary complex consisting of apo AC5, stimulatory G alpha(olf), and inhibitory G alpha(il) is investigated via classical molecular dynamics simulations. Trajectories of inhibited and stimulated binary complexes, ACS:G alpha(il) and ACS:G alpha(olf), respectively, as well as Ga-free ACS were also obtained to compare the sampled ACS conformation in the ternary complex to -those sampled under different G alpha conditions. This comparison suggests that association of both Ga subunits results in an ACS conformation similar to that sampled by the ACS:G alpha(il) complex, indicating that the ternary complex mainly samples an inactive conformation.

Water oxidation in the early steps of natural photosynthesis is fulfilled by photosystem II, which is a protein complex embedded in the thylakoid membrane inside chloroplasts. The water oxidation reaction occurs in the catalytic core of photosystem II, which consists of a Mn4Ca metal cluster, at which, after the accumulation of four oxidising equivalents through five steps (S0-S4) of the Kok-Joliot cycle, two water molecules are split into electrons, protons, and molecular oxygen. In recent years, by combining experimental and theoretical approaches, new insights have been achieved into the structural and electronic properties of different steps of the catalytic cycle. Nevertheless, the exact catalytic mechanism, especially concerning the final stages of the cycle, remains elusive and greatly debated. Herein, by means of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations, from the structural, electronic, and magnetic points of view, the S

Cholophylls are at the basis of the photosynthetic energy conversion mechanisms in algae, plants, and cyanobacteria. In photosystem II, the photoproduced electrons leave a special pair of chlorophylls (namely, P(D1) and P(D2)) that becomes cationic. This oxidizing pair [P(D1),P(D2)](+), in turn, triggers a cascade of oxidative events, eventually leading to water splitting and oxygen evolution. In the present work, using quantum mechanics/molecular mechanics calculations, we investigate the electronic structure and the dynamics of the P(D1)P(D2) special pair in both its oxidized and reduced states. In agreement with previously reported static calculations, the symmetry between the two chlorophylls was found to be broken, the positive charge being preferentially located on P(D1). Nevertheless, this study reveals for the first time that large charge fluctuations occur along dynamics, temporarily inverting the charge preference for the two branches. Finally, a vibrational analysis pinpointed that such charge fluctuations are strongly coupled to specific modes of the special pair.

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5'-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the "active site" pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions.

A detailed knowledge of the structures of the catalytic steps along the Kok-Joliot cycle of Photosystem II may help to understand the strategies adopted by this unique enzyme to achieve water oxidation. Vibrational spectroscopy has probed in the last decades the intermediate states of the catalytic cycle, although the interpretation of the data turned out to be often problematic. In the present work we use QM/MM molecular dynamics on the S-2 state to obtain the vibrational density of states at room temperature. To help the interpretation of the computational and experimental data we propose a decomposition of the Mn4CaO5 moiety into five separate parts, composed by "diamond" motifs involving four atoms. The spectral signatures arising from this analysis can be easily interpreted to assign experimentally known bands to specific molecular motions. In particular, we focused in the low frequency region of the vibrational spectrum of the S-2 state. We can therefore identify the observed bands around 600-620 cm(-1) as characteristic for the stretching vibrations involving Mn1-O1-Mn2 or Mn3-O5 moieties. (C) 2016 Elsevier B.V. All rights reserved.

Proteins of the thioredoxin (Trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. All members of the Trx family with thiol-disulfide oxidoreductase activity contain the characteristic Cys-X-X-Cys motif in their active site. Here, using Poisson-Boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigate the catalytic mechanism of DsbL, the most oxidizing Trx-like protein known to date. We observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. These results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. Intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. A consistent catalytic mechanism for DsbL, probably conferrable to other proteins of the same class, is presented. Our results suggest a functional role of hydration entropy of active-site groups.

A single amino acid exchange between the major histocompatibility complex molecules HLA-B*2705 and HLA-B*2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pKa calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in pΩ, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B*2705 complexes by ∼5 and ∼27 kJ/mol, respectively, in comparison with B*2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide- and subtype-dependent manner. In B*2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by ∼24 kJ/mol as compared with B*2705/pVIPR. B*2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide- and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype.

The characterization of the protein folding process represents one of the major challenges in molecular biology. Here, a method to simulate the folding process of a protein to its native state is reported, the essential dynamics sampling (EDS) method, and is successfully applied to detecting the correct folding pathways of two small proteins, the all-β SH3 domain of Src tyrosine kinase transforming protein (SH3) and the α/β B1 domain of streptococcal protein G (GB1). The main idea of the method is that a subset of the natural modes of fluctuation in the native state is key in directing the folding process. A biased molecular dynamics simulation is performed, in which the restrained degrees of freedom are chosen among those obtained by a principal component, or essential dynamics, analysis of the positional fluctuations of the Cα atoms in the native state. Successful folding is obtained if the restraints are applied only to the eigenvectors with lowest eigenvalues, representing the most rigid quasi-constraint motions. If the essential eigenvectors, the ones accounting for most of the variance, are used, folding is not successful. These results clearly show that the eigenvectors with lowest eigenvalues contain the main mechanical information necessary to drive the folding process, while the essential eigenvectors represent the large concerted motions which can occur without folding/unfolding the protein.

The Mn4CaO5 cluster in the oxygen-evolving complex is the catalytic core of the Photosystem II (PSII) enzyme, responsible for the water splitting reaction in oxygenic photosynthesis. The role of the redox-inactive ion in the cluster has not yet been fully clarified, although several experimental data are available on Ca2+-depleted and Ca2+-substituted PSII complexes, indicating Sr2+-substituted PSII as the only modification that preserves oxygen evolution. In this work, we investigated the structural and electronic properties of the PSII catalytic core with Ca2+ replaced with Sr2+ and Cd2+ in the S2 state of the Kok−Joliot cycle by means of density functional theory and ab initio molecular dynamics based on a quantum mechanics/ molecular mechanics approach. Our calculations do not reveal significant differences between the substituted and wild-type systems in terms of geometries, thermodynamics, and kinetics of two previously identified intermediate states along the S2 to S3 transition, namely, the open cubane S2 A and closed cubane S2 B conformers. Conversely, our calculations show different pKa values for the water molecule bound to the three investigated heterocations. Specifically, for Cd-substituted PSII, the pKa value is 5.3 units smaller than the respective value in wild type Ca-PSII. On the basis of our results, we conclude that, assuming all the cations sharing the same binding site, the induced difference in the acidity of the binding pocket might influence the hydrogen bonding network and the redox levels to prevent the further evolution of the cycle toward the S3 state.

A crucial step in the mechanism for oxygen evolution in the Photosystem II complex resides in the transition from the S2 state to the S3 state of Kok–Joliot’s cycle, in which an additional water molecule binds to the cluster. On the basis of computational chemistry calculations on Photosystem II models, we propose a reorganization mechanism involving a hydroxyl (W2) and a μ2-oxo bridge (O5) that is able to link the closed cubane S2B intermediate conformer to the S3 open cubane structure. This mechanism can reconcile the apparent conflict between recently reported water exchange and electron paramagnetic resonance experiments, and theoretical studies.

Water oxidation in photosynthetic organisms occurs through the five intermediate steps S 0 – S 4 of the Kok cycle in the oxygen evolving complex of photosystem II (PSII). Along the catalytic cycle, four electrons are subsequently removed from the Mn 4 CaO 5 core by the nearby tyrosine Tyr-Z, which is in turn oxidized by the chlorophyll special pair P680, the photo-induced primary donor in PSII. Recently, two Mn 4 CaO 5 conformations, consistent with the S 2 state (namely, S 2 A and S 2 B models) were suggested to exist, perhaps playing a different role within the S 2 -to- S 3 transition. Here we report multiscale ab initio density functional theory plus U simulations revealing that upon such oxidation the relative thermodynamic stability of the two previously proposed geometries is reversed, the S 2 B state becoming the leading conformation. In this latter state a proton coupled electron transfer is spontaneously observed at ∼100 fs at room temperature dynamics. Upon oxidation, the Mn cluster, which is tightly electronically coupled along dynamics to the Tyr-Z tyrosyl group, releases a proton from the nearby W1 water molecule to the close Asp-61 on the femtosecond timescale, thus undergoing a conformational transition increasing the available space for the subsequent coordination of an additional water molecule. The results can help to rationalize previous spectroscopic experiments and confirm, for the first time to our knowledge, that the water-splitting reaction has to proceed through the S 2 B conformation, providing the basis for a structural model of the S 3 state.

An important approach in the design of new environmentally friendly materials is represented by the study of analogous systems already existing in nature. In the search for new water splitting catalysts, the corresponding natural analogue is represented by the oxygen-evolving complex of photosystem II, which is a large membrane protein complex present in photosynthetic organisms. The understanding of the catalytic strategy of its active Mn4CaO5 core is important to unravel the mechanisms of water oxidation in photosynthesis and can serve as an inspiring model for the design of biomimetic catalysts based on largely non-toxic, earth abundant elements. The magnetic interactions between Mn ions are studied in the present work by means of DFT+U broken symmetry ab initio molecular dynamics within a quantum mechanics/molecular mechanics framework. The room temperature dynamics of two different structural models (i.e. with total high-spin and total low-spin ground states) was stable during the simulated time. We observed large fluctuations of the magnetic coupling

Trans-solubleN-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complexes formed between the SNARE motifs of synaptobrevin II, SNAP-25, and syntaxin play an essential role in Ca2+-regulated exocytosis. Apart from the well studied interactions of the SNARE domains, little is known about the functional relevance of other evolutionarily conserved structures in the SNARE proteins. Here, we show that substitution of two highly conserved tryptophan residues within the juxtamembrane domain (JMD) of the vesicular SNARE Synaptobrevin II (SybII) profoundly impairs priming of granules in mouse chromaffin cells without altering catecholamine release from single vesicles. Using molecular dynamic simulations of membrane-embedded SybII, we show that Trp residues of the JMD influence the electrostatic surface potential by controlling the position of neighboring lysine and arginine residues at the membrane–water interface. Our observations indicate a decisive role of the tryptophan moiety of SybII in keeping the vesicles in the release-ready state and support a model wherein tryptophan-mediated protein–lipid interactions assist in bridging the apposing membranes before fusion.

Major histocompatibility complex (MHC) class I proteins are expressed on the cell surface where they present foreign and self-peptides to effector cells of the immune system. While an understanding of the structural prerequisites for antigen presentation has already been achieved, insight into subtype- or peptide-dependent dynamical characteristics of a peptide–MHC antigen is so far largely obscure. We approached this problem by employing 400-ns molecular dynamics simulations with two human MHC class I subtypes as model systems: the ankylosing spondylitis-associated HLA-B∗27:05 and the non-ankylosing spondylitis-associated HLA-B∗27:09. Both proteins differ only by a micropolymorphism at the floor of the peptide binding groove (Asp116His). A viral (pLMP2) and three self-peptides (pVIPR, pGR, and TIS) were evaluated. The stability of the binding grooves was found to be both subtype dependent and peptide dependent. A detachment from the C- and/or N-terminal pockets was observed for all peptides except TIS, resulting in a stabilization of the α1-helix in both TIS-displaying subtypes. Estimates of the entropy associated with the bound peptides showed an increased entropy for pLMP2 presented by B∗27:05 as compared to B∗27:09, in contrast to the self-peptides. Additionally, the flexibility of the α1-helix that is probably important for receptor binding to the B27:peptide epitope is significantly enhanced for B∗27:05. These in silico results show that the dynamic properties of peptide–MHC complexes are affected both by the bound peptide and by micropolymorphisms of the heavy chain. Our findings suggest a role for the conformational flexibility of MHC class I molecules in the context of recognition by receptors on effector cells.

Human leukocyte antigen (HLA) class I molecules consist of a heavy chain, β2-microglobulin, and a peptide that are noncovalently bound. Certain HLA-B27 subtypes are associated with ankylosing spondylitis (such as HLA-B*2705), whereas others (such as HLA-B*2709) are not. Both differ in only one residue (Asp116 and His116, respectively) in the F pocket that accommodates the peptide C-terminus. An isotope-edited IR spectroscopy study of these HLA-B27 subtypes complexed with the self-peptide RRKWRRWHL was carried out, revealing that the heavy chain is more flexible in the HLA-B*2705 than in the HLA-B*2709 subtype. In agreement with these experimental data, molecular dynamics simulations showed an increased flexibility of the HLA-B*2705 binding groove in comparison with that of the HLA-B*2709 subtype. This difference correlates with an opening of the HLA-B*2705 binding groove, accompanied by a partial detachment of the C-terminal peptide anchor. These combined results demonstrate how the deeply embedded polymorphic heavy-chain residue 116 influences the flexibility of the peptide binding groove in a subtype-dependent manner, a feature that could also influence the recognition of the HLA-B27 complexes by effector cells.

Light harvesting from the Sun by antenna complexes surrounding the reaction center of Photosystem II represents the first step of the natural oxygenic photosynthesis performed by plants, algae and cyanobacteria. The excitation energy derived from the sunlight is absorbed by the chlorophylls of the antenna and subsequently conveyed to the reaction center of Photosystem II through resonant energy transfer mechanisms. In the special pair of chlorophylls of the reaction center the charge separation occurs, eventually leading to the oxidation of water molecules into protons, electrons and molecular oxygen. The adsorption properties of the antenna chlorophylls are ad hoc modulated by the protein environment to guarantee fast energy transfer and minimize side and back reactions. At the same time these properties are influenced by the molecular fluctuations occurring at physiological temperature. In the present work, combining classical molecular dynamics simulations with the Charge Density Coupling method, we estimated the impact of the thermal fluctuations on the site energy shift of the chlorophylls embedded in the Photosystem II complex. Our results show how the effect of the molecular fluctuations is not homogeneous throughout the complex, although the symmetry of the homodimer is maintained. Many peripheral chromophores undergo fluctuations larger then 10 kJ/mol around the average values. Possible physiological roles of such fluctuations are discussed.

Water oxidation in the early steps of natural photosynthesis is fulfilled by photosystem II, which is a protein complex embedded in the thylakoid membrane inside chloroplasts. The water oxidation reaction occurs in the catalytic core of photosystem II, which consists of a Mn4Ca metal cluster, at which, after the accumulation of four oxidising equivalents through five steps (S0-S4) of the Kok-Joliot cycle, two water molecules are split into electrons, protons, and molecular oxygen. In recent years, by combining experimental and theoretical approaches, new insights have been achieved into the structural and electronic properties of different steps of the catalytic cycle. Nevertheless, the exact catalytic mechanism, especially concerning the final stages of the cycle, remains elusive and greatly debated. Herein, by means of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations, from the structural, electronic, and magnetic points of view, the S3 state before and upon oxidation has been characterised. In contrast with the S2 state, the oxidation of the S3 state is not followed by a spontaneous proton-coupled electron-transfer event. Nevertheless, upon modelling the reduction of the tyrosine residue in photosystem II (TyrZ ) and the protonation of Asp61, spontaneous proton transfer occurs, leading to the deprotonation of an oxygen atom bound to Mn1; thus making it available for O-O bond formation.

Photosynthetic water oxidation is catalyzed by the Mn4Ca cluster in photosystem II (PSII). The nearby redox-active tyrosine (Y-Z) serves as a direct electron acceptor of the Mn4Ca cluster and it forms a low-barrier H-bond (LBHB) with a neighboring histidine residue (D1-His190). Experimental evidence indicates that Y-Z oxidation triggers changes in the hydrogen bonding network that precede proton abstraction from the Mn4Ca cluster. In order to characterize such changes, we compare ab initio molecular dynamics simulations of different states of the catalytic cycle of PSII with dynamics of isolated tyrosine models (namely, p-cresol) in different oxidation states. The systematic comparison of the H-bond networks in different simulated systems suggests that the Y-Z oxidation leads to a water hydration pattern which is more similar to that of the neutral p-cresol rather than that of the p-cresol anion. Our simulations also reveal the twofold nature of the interactions between Y-Z and the Mn4Ca cluster. Firstly, the Y-Z oxidation triggers rapid structural changes of the H-bond pattern in the proximity of the cluster which have been observed to propagate on the ps time scale on the Ca2+ hydration shell up to other water molecules in the proximity of the cluster. Secondly, it is clear that Y-Z interacts with the Mn4Ca cluster also through Coulombic interactions mediated by CP43-Arg357 through the remaining positive charge of the pair. Our results are able to identify, for the first time, the structural rearrangements guided by the oxidation of Y-Z necessary for the evolution of the water splitting reaction in PSII. Based on these findings, we propose a mechanism of structural changes which is functional towards the progression of the catalytic cycle in PSII.

The Mn4CaO5 cluster of photosystem II promotes a crucial step in the oxygenic photosynthesis, namely, the water-splitting reaction. The structure of such cluster in the S-1 state of the Kok-Joliot's cycle has been recently resolved by femtosecond X-ray free-electron laser (XFEL) measurements. However, the XFEL results are characterized by appreciable discrepancies with previous X-ray diffraction (XRD), as well as with S-1 models based on ab initio calculations. We provide here a unifying picture based on a combined set of DFT-based structures and molecular dynamics simulations of the S-0 and S-1 states. Our findings indicate that the XFEL results cannot be interpreted on the grounds of a single structure. A combination of two S-1 stable isomers together with a minority contribution of the S-0 state is necessary to reproduce XFEL results within 0.16 angstrom.