Predicting the Structure of Enzymes with Metal Cofactors: The Example of [FeFe] Hydrogenases.

The advent of deep learning algorithms for protein folding opened a new era in the ability of predicting and optimizing the function of proteins once the sequence is known. The task is more intricate when cofactors like metal ions or small ligands are essential to functioning. In this case, the combined use of traditional simulation methods based on interatomic force fields and deep learning predictions is mandatory. We use the example of [FeFe] hydrogenases, enzymes of unicellular algae promising for biotechnology applications to illustrate this situation. [FeFe] hydrogenase is an iron-sulfur protein that catalyzes the chemical reduction of protons dissolved in liquid water into molecular hydrogen as a gas. Hydrogen production efficiency and cell sensitivity to dioxygen are important parameters to optimize the industrial applications of biological hydrogen production. Both parameters are related to the organization of iron-sulfur clusters within protein domains. In this work, we propose possible three-dimensional structures of Chlorella vulgaris 211/11P [FeFe] hydrogenase, the sequence of which was extracted from the recently published genome of the given strain. Initial structural models are built using: (i) the deep learning algorithm AlphaFold; (ii) the homology modeling server SwissModel; (iii) a manual construction based on the best known bacterial crystal structure. Missing iron-sulfur clusters are included and microsecond-long molecular dynamics of initial structures embedded into the water solution environment were performed. Multiple-walkers metadynamics was also used to enhance the sampling of structures encompassing both functional and non-functional organizations of iron-sulfur clusters. The resulting structural model provided by deep learning is consistent with functional [FeFe] hydrogenase characterized by peculiar interactions between cofactors and the protein matrix.

Amyloid-ß Tetramers and Divalent Cations at the Membrane/Water Interface: Simple Models Support a Functional Role.

Charge polarization at the membrane interface is a fundamental process in biology. Despite the lower concentration compared to the abundant monovalent ions, the relative abundance of divalent cations (Ca2+, Mg2+, Zn2+, Fe2+, Cu2+) in particular spaces, such as the neuron synapse, raised many questions on the possible effects of free multivalent ions and of the required protection of membranes by the eventual defects caused by the free forms of the cations. In this work, we first applied a recent realistic model of divalent cations to a well-investigated model of a polar lipid bilayer, di-myristoyl phosphatidyl choline (DMPC). The full atomistic model allows a fairly good description of changes in the hydration of charged and polar groups upon the association of cations to lipid atoms. The lipid-bound configurations were analyzed in detail. In parallel, amyloid-ß 1-42 (Aß42) peptides assembled into tetramers were modeled at the surface of the same bilayer. Two of the protein tetramers' models were loaded with four Cu2+ ions, the latter bound as in DMPC-free Aß42 oligomers. The two Cu-bound models differ in the binding topology: one with each Cu ion binding each of the monomers in the tetramer; one with pairs of Cu ions linking two monomers into dimers, forming tetramers as dimers of dimers. The models here described provide hints on the possible role of Cu ions in synaptic plasticity and of Aß42 oligomers in storing the same ions away from lipids. The release of structurally disordered peptides in the synapse can be a mechanism to recover ion homeostasis and lipid membranes from changes in the divalent cation concentration.

Probing Reactivity with External Forces: The Case of Nitroacetamides in Water.

Many computational methods have been applied to interpret and predict changes in reactivity by slight modifications of a given molecular scaffold. We describe a novel and simple method based on approximate density-functional theory of valence electrons that can be applied within a large high-performance computational infrastructure to probe such changes using a statistical sample of molecular configurations, including the solvent. All the used computational tools are fully open-source. Following our previous application, we are able to explain the high acidity of C-H bond at α position in nitro compounds when the amide linkage an ammonium group is inserted into the α substituent.

Modelling Protein Plasticity: The Example of Frataxin and Its Variants.

Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the FXN gene lead to reduced production of functional frataxin, with the consequent dyshomeostasis of iron. FXN variants are at the basis of neurological impairment (the Friedreich's ataxia) and several types of cancer. By using altruistic metadynamics in conjunction with the maximal constrained entropy principle, we estimate the change of free energy in the protein unfolding of frataxin and of some of its pathological mutants. The sampled configurations highlight differences between the wild-type and mutated sequences in the stability of the folded state. In partial agreement with thermodynamic experiments, where most of the analyzed variants are characterized by lower thermal stability compared to wild type, the D104G variant is found with a stability comparable to the wild-type sequence and a lower water-accessible surface area. These observations, obtained with the new approach we propose in our work, point to a functional switch, affected by single-point mutations, of frataxin from iron storage to iron release. The method is suitable to investigate wide structural changes in proteins in general, after a proper tuning of the chosen collective variable used to perform the transition.

Measuring Shared Electrons in Extended Molecular Systems: Covalent Bonds from Plane-Wave Representation of Wave Function.

In the study of materials and macromolecules by first-principle methods, the bond order is a useful tool to represent molecules, bulk materials and interfaces in terms of simple chemical concepts. Despite the availability of several methods to compute the bond order, most applications have been limited to small systems because a high spatial resolution of the wave function and an all-electron representation of the electron density are typically required. Both limitations are critical for large-scale atomistic calculations, even within approximate density-functional theory (DFT) approaches. In this work, we describe our methodology to quickly compute delocalization indices for all atomic pairs, while keeping the same representation of the wave function used in most compute-intensive DFT calculations on high-performance computing equipment. We describe our implementation into a post-processing tool, designed to work with Quantum ESPRESSO, a popular open-source DFT package. In this way, we recover a description in terms of covalent bonds from a representation of wave function containing no explicit information about atomic types and positions.

Computational models explain how copper binding to amyloid-ß peptide oligomers enhances oxidative pathways.

Amyloid-ß (Aß) peptides are intrinsically disordered peptides and their aggregation is the major hallmark of Alzheimer's disease (AD) development. The interactions between copper ions and Aß peptides create catalysts that activate the production of reactive oxygen species in the synaptic region, a reactivity that is strongly related to AD onset. Recent experimental work [Gu et al., Sci. Rep., 2018, 8(1), 16190] confirmed that the oxidative reactivity of Cu-Aß catalyzes the formation of Tyr-Tyr crosslinks in peptide dimers. This work provides a structural basis to these observations, describing structures of Cu-Aß dimers that enhance the propagation of the oxidative pathways activated around the Cu center. Among these, the formation of Tyr-Tyr crosslinks becomes more likely when previous crosslinks involve Cu forming bridges between different peptides. Peptides are, therefore, easily assembled into dimers and tetramers, the latter being dimers of dimers. The size of such dimers and tetramers fits with ion mobility mass spectrometry results [Sitkiewicz et al., J. Mol. Biol., 2014, 426(15), 2871].

Towards High-Throughput Modelling of Copper Reactivity Induced by Structural Disorder in Amyloid Peptides.

Transition metal ions often interact with disordered proteins. The affinity is high enough to compete with structured proteins, but the catalytic activity of the metal centre is often out of control and, therefore, potentially dangerous for cells. An example is a single copper ion interacting with the amyloid-ß (Aß) peptide and triplet dioxygen, an interaction that is fundamental in producing reactive oxygen species in neurodegeneration. High-throughput modelling of the Cu-Aß-O2 system was performed with the aim of providing a tool to dissect the structural features that characterise dangerous Cu-based catalysts in neurodegeneration. This study showed that the production of superoxide is a process with low-energy intermediate species, once a small population of high-energy CuI -Aß complex is formed. This population is enhanced when Cu bridges two different peptides in 1:1 Cu:Aß dimers. Despite the bias for high-energy reduced reactant species, the reduction of CuII -Aß product by superoxide can also occur, in addition to that by ascorbate, because the structural disorder produces a small population of oxidant species characterised by unstable CuII coordination, coexisting with the most abundant reductant species, characterised by stable CuII coordination.

Multi-scale theoretical approach to X-ray absorption spectra in disordered systems: an application to the study of Zn(ii) in water.

We develop a multi-scale theoretical approach aimed at calculating from first principles X-ray absorption spectra of liquid solutions and disordered systems. We test the method by considering the paradigmatic case of Zn(ii) in water which, besides being relevant in itself, is also of interest for biology. With the help of classical molecular dynamics simulations we start by producing bunches of configurations differing for the Zn(ii)-water coordination mode. Different coordination modes are obtained by making use of the so-called dummy atoms method. From the collected molecular dynamics trajectories, snapshots of a more manageable subsystem encompassing the metal site and two solvation layers are cut out. Density functional theory is used to optimize and relax these reduced system configurations employing a uniform dielectric to mimic the surrounding bulk liquid water. On the resulting structures, fully quantum mechanical X-ray absorption spectra calculations are performed by including core-hole effects and core-level shifts. The proposed approach does not rely on any guessing or fitting of the force field or of the atomic positions of the system. The comparison of the theoretically computed spectrum with the experimental Zn K-edge XANES data unambiguously demonstrates that among the different a priori possible geometries, Zn(ii) in water lives in an octahedral coordination mode.

Understanding the Exceptional Properties of Nitroacetamides in Water: A Computational Model Including the Solvent.

Proton transfer in water involving C⁻H bonds is a challenge and nitro compounds have been studied for many years as good examples. The effect of substituents on acidity of protons geminal to the nitro group is exploited here with new p K a measurements and electronic structure models, the latter including explicit water environment. Substituents with the amide moiety display an exceptional combination of acidity and solubility in water. In order to find a rationale for the unexpected p K a changes in the (ZZ ' )NCO- substituents, we measured and modeled the p K a with Z=Z ' =H and Z=Z ' =methyl. The dominant contribution to the observed p K a can be understood with advanced computational experiments, where the geminal proton is smoothly moved to the solvent bath. These models, mostly based on density-functional theory (DFT), include the explicit solvent (water) and statistical thermal fluctuations. As a first approximation, the change of p K a can be correlated with the average energy difference between the two tautomeric forms (aci and nitro, respectively). The contribution of the solvent molecules interacting with the solute to the proton transfer mechanism is made evident.

Modeling (15)N NMR chemical shift changes in protein backbone with pressure.

Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.

Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)-Aß Peptide and O2.

Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid-ß (Aß) is found in AD brains, and Cu-Aß could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HO˙ in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aß-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aß in the presence of ascorbate occurs mainly via a free O2˙(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aß, and opens the possibility that Cu-Aß-catalyzed O2˙(-) contributes to oxidative stress in AD, and hence may be of interest.

Metal ions and intrinsically disordered proteins and peptides: from Cu/Zn amyloid-ß to general principles.

The interaction of d-block metal ions (Cu, Zn, Fe, etc.) with intrinsically disordered proteins (IDPs) has gained interest, partly due to their proposed roles in several diseases, mainly neurodegenerative. A prominent member of IDPs is the peptide amyloid-ß (Aß) that aggregates into metal-enriched amyloid plaques, a hallmark of Alzheimer's disease, in which Cu and Zn are bound to Aß. IDPs are a class of proteins and peptides that lack a unique 3D structure when the protein is isolated. This disordered structure impacts their interaction with metal ions compared with structured metalloproteins. Metalloproteins either have a preorganized metal binding site or fold upon metal binding, resulting in defined 3D structure with a well-defined metal site. In contrast, for Aß and likely most of the other IDPs, the affinity for Cu(I/II) and Zn(II) is weaker and the interaction is flexible with different coordination sites present. Coordination of Cu(I/II) with Aß is very dynamic including fast Cu-exchange reactions (milliseconds or less) that are intrapeptidic between different sites as well as interpeptidic. This highly dynamic metal-IDP interaction has a strong impact on reactivity and potential biological role: (i) Due to the low affinity compared with classical metalloproteins, IDPs likely bind metals only at special places or under special conditions. For Aß, this is likely in the neurons that expel Zn or Cu into the synapse and upon metal dysregulation occurring in Alzheimer's disease. (ii) Amino acid substitutions (mutations) on noncoordinating residues can change drastically the coordination sphere. (iii) Considering the Cu/Zn-Aß aberrant interaction, therapeutic strategies can be based on removal of Cu/Zn or precluding their binding to the peptide. The latter is very difficult due to the multitude of metal-binding sites, but the fast koff facilitates removal. (iv) The high flexibility of the Cu-Aß complex results in different conformations with different redox activity. Only some conformations are able to produce reactive oxygen species. (v) Other, more specific catalysis (like enzymes) is very unlikely for Cu/Zn-Aß. (vi) The Cu/Zn exchange reactions with Aß are faster than the aggregation process and can hence have a strong impact on this process. In conclusion, the coordination chemistry is fundamentally different for most of IDPs compared with the classical, structured metalloproteins or with (bio)-inorganic complexes. The dynamics is a key parameter to understand this interaction and its potential biological impact.

Dioxygen activation in the Cu-amyloid ß complex.

We investigate, by means of density-functional theory, the binding of dioxygen to Cu(I)-amyloid ß (Aß), one of the first steps in the oxidation of ascorbate by dioxygen. Cu, Aß, ascorbate and dioxygen are all present in the synapse during neurodegeneration, when the above species can trigger an irreversible oxidative stress inducing the eventual death of neurons. The binding of dioxygen to Cu(I) is possible and its role in dioxygen activation of Cu ligands and of residues in the first coordination sphere is described in atomic detail. Dioxygen is activated when a micro-environment suitable for a square-planar Cu(2+) coordination is present and a negatively charged group like Asp 1 carboxylate takes part in the Cu coordination anti to O2.

Probing protein stability: towards a computational atomistic, reliable, affordable, and improvable model.

We present an improved application of a recently proposed computational method designed to evaluate the change of free energy as a function of the average value of a suitably chosen collective variable in proteins. The method is based on a full atomistic description of the protein and its environment. The goal is to understand how the protein melting temperature changes upon single-point mutations, because the sign of the temperature variation will allow us to discriminate stabilizing vs. destabilizing mutations in protein sequences. In this refined application the method is based on altruistic well-tempered metadynamics, a variant of multiple-walkers metadynamics. The resulting metastatistics is then modulated by the maximal constrained entropy principle. The latter turns out to be especially helpful in free-energy calculations as it is able to alleviate the severe limitations of metadynamics in properly sampling folded and unfolded configurations. In this work we apply the computational strategy outlined above in the case of the bovine pancreatic trypsin inhibitor, a well-studied small protein, which is a reference for computer simulations since decades. We compute the variation of the melting temperature characterizing the folding-unfolding process between the wild-type protein and two of its single-point mutations that are seen to have opposite effect on the free energy changes. The same approach is used for free energy difference calculations between a truncated form of frataxin and a set of five of its variants. Simulation data are compared to in vitro experiments. In all cases the sign of the change of melting temperature is reproduced, under the further approximation of using an empirical effective mean-field to average out protein-solvent interactions.

The mechanism of hydrogen uptake in [NiFe] hydrogenase: first-principles molecular dynamics investigation of a model compound.

The recent discovery of a model compounds of [NiFe] hydrogenase that catalyzes the heterolytic cleavage of the H(2) molecule into a proton and a stable hydride in water solution under room conditions opened up the possibility to understand the mechanism of H(2) uptake by this peculiar class of enzymes. The simplest model compound belongs to the class of NiRu bimetallic cationic complexes mimicking, in water solution and at room conditions, the hydrogenase active site. By using first-principles molecular dynamics computer simulations, in the Car-Parrinello scheme, we investigated models including the water solvent and nitrate counterions. Several simulations, starting from different initial configurations, provided information on the first step of the H(2) cleavage: (1) the pathway of H(2) approach towards the active site; (2) the role of the ruthenium-bonded water molecule in providing a base that extracts the proton from the activated H(2) molecule; (3) the minor role of Ni in activating the H(2) molecule and its role in stabilizing the hydride produced.

Aggregates Sealed by Ions.

The chapter draws a line connecting some recent results where the role of ions is found essential in sealing more or less pre-organized assemblies of macromolecules. We draw some dots along the line that starts from the effect of the ionic atmosphere and ends with the chemical bonds formed by multivalent ions acting as bridges between macromolecules. Many of these dots involve structurally disordered peptides and disordered regions of proteins. A broad perspective of the role of multivalent ions in assisting the assembly process, shifting population in polymorphic states, and sealing protein aggregates, is suggested.

Amyloid ß Dodecamer Disrupts the Neuronal Membrane More Strongly than the Mature Fibril: Understanding the Role of Oligomers in Neurotoxicity.

The amyloid cascade hypothesis states that senile plaques, composed of amyloid ß (Aß) fibrils, play a key role in Alzheimer's disease (AD). However, recent experiments have shown that Aß oligomers are more toxic to neurons than highly ordered fibrils. The molecular mechanism underlying this observation remains largely unknown. One of the possible scenarios for neurotoxicity is that Aß peptides create pores in the lipid membrane that allow Ca2+ ions to enter cells, resulting in a signal of cell apoptosis. Hence, one might think that oligomers are more toxic due to their higher ability to create ion channels than fibrils. In this work, we study the effect of Aß42 dodecamer and fibrils on a neuronal membrane, which is similar to that observed in AD patients, using all-atom molecular dynamics simulations. Due to short simulation times, we cannot observe the formation of pores, but useful insight on the early events of this process has been obtained. Namely, we showed that dodecamer distorts the lipid membrane to a greater extent than fibrils, which may indicate that ion channels can be more easily formed in the presence of oligomers. Based on this result, we anticipate that oligomers are more toxic than mature fibrils, as observed experimentally. Moreover, the Aß-membrane interaction was found to be governed by the repulsive electrostatic interaction between Aß and the ganglioside GM1 lipid. We calculated the bending and compressibility modulus of the membrane in the absence of Aß and obtained good agreement with the experiment. We predict that the dodecamer will increase the compressibility modulus but has little effect on the bending modulus. Due to the weak interaction with the membrane, fibrils insignificantly change the membrane elastic properties.

Exploring the reactions of ß-amyloid (Aß) peptide 1-28 with Al(III) and Fe(III) ions.

The reactions of human ß-amyloid peptide 1-28 (Aß28) with Al(III) and Fe(III) ions were investigated by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) under pH conditions close to physiological ones. (1)H NMR titrations, performed in the 5.3-8.0 pH range, revealed that no measurable amounts of Aß28-Al(III) or Aß28-Fe(III) adducts are formed; such metal adducts could not be obtained even by changing a number of experimental conditions, e.g., temperature, buffer, nature of the salt, etc. These observations were later confirmed by ESI-MS. It is thus demonstrated that Aß28, at physiological pH, is not able to form binary complexes with Al(III) and Fe(III) ions of sufficient stability to compete with metal hydroxide precipitation. The biological implications of these findings are discussed in the frame of current literature.

Polyphenols as Potential Metal Chelation Compounds Against Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease affecting more than 50 million people worldwide. The pathology of this multifactorial disease is primarily characterized by the formation of amyloid-ß (Aß) aggregates; however, other etiological factors including metal dyshomeostasis, specifically copper (Cu), zinc (Zn), and iron (Fe), play critical role in disease progression. Because these transition metal ions are important for cellular function, their imbalance can cause oxidative stress that leads to cellular death and eventual cognitive decay. Importantly, these transition metal ions can interact with the amyloid-ß protein precursor (AßPP) and Aß42 peptide, affecting Aß aggregation and increasing its neurotoxicity. Considering how metal dyshomeostasis may substantially contribute to AD, this review discusses polyphenols and the underlying chemical principles that may enable them to act as natural chelators. Furthermore, polyphenols have various therapeutic effects, including antioxidant activity, metal chelation, mitochondrial function, and anti-amyloidogenic activity. These combined therapeutic effects of polyphenols make them strong candidates for a moderate chelation-based therapy for AD.

Zn-Induced Interactions Between SARS-CoV-2 orf7a and BST2/Tetherin.

We present in this work a first X-ray Absorption Spectroscopy study of the interactions of Zn with human BST2/tetherin and SARS-CoV-2 orf7a proteins as well as with some of their complexes. The analysis of the XANES region of the measured spectra shows that Zn binds to BST2, as well as to orf7a, thus resulting in the formation of BST2-orf7a complexes. This structural information confirms the the conjecture, recently put forward by some of the present Authors, according to which the accessory orf7a (and possibly also orf8) viral protein are capable of interfering with the BST2 antiviral activity. Our explanation for this behavior is that, when BST2 gets in contact with Zn bound to the orf7a Cys15 ligand, it has the ability of displacing the metal owing to the creation of a new disulfide bridge across the two proteins. The formation of this BST2-orf7a complex destabilizes BST2 dimerization, thus impairing the antiviral activity of the latter.