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.

When Metal Complexes Evolve, and a Minor Species Is the Most Active: the Case of Bis(Phenanthroline)Copper in the Catalysis of Glutathione Oxidation and Hydroxyl Radical Generation.

Several copper-ligands, including 1,10-phenanthroline (Phen), have been investigated for anticancer purposes based on their capacity to bind excess copper (Cu) in cancer tissues and form redox active complexes able to catalyse the formation of reactive oxygen species (ROS), ultimately leading to oxidative stress and cell death. Glutathione (GSH) is a critical compound as it is highly concentrated intracellularly and can reduce and dissociate copper(II) from the ligand forming poorly redox-active copper(I)-thiolate clusters. Here we report that Cu-Phen2  speciation evolves in physiologically relevant GSH concentrations. Experimental and computational experiments suggest that at pH 7.4 mostly copper(I)-GSH clusters are formed, but a minor species of copper(I) bound to one Phen and forming ternary complexes with GSH (GS-Cu-Phen) is the redox active species, oxidizing quite efficiently GSH to GSSG and forming HO• radicals. This minor active species becomes more populated at lower pH, such as typical lysosomal pH 5, resulting in faster GSH oxidation and HO• production. Consistently, cell culture studies showed lower toxicity of Cu-Phen2 upon inhibition of lysosomal acidification. Overall, this study underscores that sub-cellular localisation can considerably influence the speciation of Cu-based drugs and that minor species can be the most redox- and biologically- active.

Chasing the Elusive "In-Between" State of the Copper-Amyloid ß Complex by X-ray Absorption through Partial Thermal Relaxation after Photoreduction.

The redox activity of Cu ions bound to the amyloid-ß (Aß) peptide is implicated as a source of oxidative stress in the context of Alzheimer's disease. In order to explain the efficient redox cycling between CuII -Aß (distorted square-pyramidal) and CuI -Aß (digonal) resting states, the existence of a low-populated "in-between" state, prone to bind Cu in both oxidation states, has been postulated. Here, we exploited the partial X-ray induced photoreduction at 10 K, followed by a thermal relaxation at 200 K, to trap and characterize by X-ray Absorption Spectroscopy (XAS) a partially reduced Cu-Aß1-16 species different from the resting states. Remarkably, the XAS spectrum is well-fitted by a previously proposed model of the "in-between" state, hence providing the first direct spectroscopic characterization of an intermediate state. The present approach could be used to explore and identify the catalytic intermediates of other relevant metal complexes.

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.

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.

Single-Nucleotide Polymorphism of PPARγ, a Protein at the Crossroads of Physiological and Pathological Processes.

Genome polymorphisms are responsible for phenotypic differences between humans and for individual susceptibility to genetic diseases and therapeutic responses. Non-synonymous single-nucleotide polymorphisms (nsSNPs) lead to protein variants with a change in the amino acid sequence that may affect the structure and/or function of the protein and may be utilized as efficient structural and functional markers of association to complex diseases. This study is focused on nsSNP variants of the ligand binding domain of PPARγ a nuclear receptor in the superfamily of ligand inducible transcription factors that play an important role in regulating lipid metabolism and in several processes ranging from cellular differentiation and development to carcinogenesis. Here we selected nine nsSNPs variants of the PPARγ ligand binding domain, V290M, R357A, R397C, F360L, P467L, Q286P, R288H, E324K, and E460K, expressed in cancer tissues and/or associated with partial lipodystrophy and insulin resistance. The effects of a single amino acid change on the thermodynamic stability of PPARγ, its spectral properties, and molecular dynamics have been investigated. The nsSNPs PPARγ variants show alteration of dynamics and tertiary contacts that impair the correct reciprocal positioning of helices 3 and 12, crucially important for PPARγ functioning.

Computational and experimental studies on ß-sheet breakers targeting Aß1-40 fibrils.

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aß1-40 peptide in water in interaction with short peptides (ß-sheet breakers) mimicking the 17-21 region of the Aß1-40 sequence. Various systems differing in the customized ß-sheet breaker structure have been studied. Specifically we have considered three kinds of ß-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that ß-sheet breakers are able to inhibit in vitro fibril formation and prevent the ß sheet folding of portions of the Aß1-40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 ß-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aß1-40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aß1-40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17-21 Aß1-40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 ß-sheet breaker.

Mesoscopic behavior from microscopic Markov dynamics and its application to calcium release channels.

A major challenge in biology is to understand how molecular processes determine phenotypic features. We address this fundamental problem in a class of model systems by developing a general mathematical framework that allows the calculation of mesoscopic properties from the knowledge of microscopic Markovian transition probabilities. We show how exact analytic formulae for the first and second moments of resident time distributions in mesostates can be derived from microscopic resident times and transition probabilities even for systems with a large number of microstates. We apply our formalism to models of the inositol trisphosphate receptor, which plays a key role in generating calcium signals triggering a wide variety of cellular responses. We demonstrate how experimentally accessible quantities, such as opening and closing times and the coefficient of variation of inter-spike intervals, and other, more elaborated, quantities can be analytically calculated from the underlying microscopic Markovian dynamics. A virtue of our approach is that we do not need to follow the detailed time evolution of the whole system, as we derive the relevant properties of its steady state without having to take into account the often extremely complicated transient features. We emphasize that our formulae fully agree with results obtained by stochastic simulations and approaches based on a full determination of the microscopic system's time evolution. We also illustrate how experiments can be devised to discriminate between alternative molecular models of the inositol trisphosphate receptor. The developed approach is applicable to any system described by a Markov process and, owing to the analytic nature of the resulting formulae, provides an easy way to characterize also rare events that are of particular importance to understand the intermittency properties of complex dynamic systems.

Copper-zinc cross-modulation in prion protein binding.

In this paper we report a systematic XAS study of a set of samples in which Cu(II) was progressively added to complexes in which Zn(II) was bound to the tetra-octarepeat portion of the prion protein. This work extends previous EPR and XAS analysis in which, in contrast, the effect of adding Zn(II) to Cu(II)-tetra-octarepeat complexes was investigated. Detailed structural analysis of the XAS spectra taken at both the Cu and Zn K-edge when the two metals are present at different relative concentrations revealed that Zn(II) and Cu(II) ions compete for binding to the tetra-octarepeat peptide by cross-regulating their relative binding modes. We show that the specific metal-peptide coordination mode depends not only, as expected, on the relative metal concentrations, but also on whether Zn(II) or Cu(II) was first bound to the peptide. In particular, it seems that the Zn(II) binding mode in the absence of Cu(II) is able to promote the formation of small peptide clusters in which triplets of tetra-octarepeats are bridged by pairs of Zn ions. When Cu(II) is added, it starts competing with Zn(II) for binding, disrupting the existing peptide cluster arrangement, despite the fact that Cu(II) is unable to completely displace Zn(II). These results may have a bearing on our understanding of peptide-aggregation processes and, with the delicate cross-regulation balancing we have revealed, seem to suggest the existence of an interesting, finely tuned interplay among metal ions affecting protein binding, capable of providing a mechanism for regulation of metal concentration in cells.

Probing the Dynamic Landscape: From Static to Time-Resolved X-Ray Absorption Spectroscopy to Investigate Copper Redox Chemistry in Neurodegenerative Disorders.

Copper (Cu), with its ability to exist in various oxidation states, notably Cu(I) and Cu(II), plays a crucial role in diverse biological redox reactions. This includes its involvement in pathways associated with oxidative stress in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Transmissible Spongiform Encephalopathies. This paper offers an overview of X-ray Absorption Spectroscopy (XAS) studies designed to elucidate the interactions between Cu ions and proteins or peptides associated with these neurodegenerative diseases. The emphasis lies on XAS specificity, revealing the local coordination environment, and on its sensitivity to Cu oxidation states. Furthermore, the paper focuses on XAS applications targeting the characterization of intermediate reaction states and explores the opportunities arising from recent advancements in time-resolved XAS at ultrabright synchrotron and Free Electron Laser radiation sources.

Revisiting the pro-oxidant activity of copper: interplay of ascorbate, cysteine, and glutathione.

Copper (Cu) is essential for most organisms, but it can be poisonous in excess, through mechanisms such as protein aggregation, trans-metallation, and oxidative stress. The latter could implicate the formation of potentially harmful reactive oxygen species (O2•-, H2O2, and HO•) via the redox cycling between Cu(II)/Cu(I) states in the presence of dioxygen and physiological reducing agents such as ascorbate (AscH), cysteine (Cys), and the tripeptide glutathione (GSH). Although the reactivity of Cu with these reductants has been previously investigated, the reactions taking place in a more physiologically relevant mixture of these biomolecules are not known. Hence, we report here on the reactivity of Cu with binary and ternary mixtures of AscH, Cys, and GSH. By measuring AscH and thiol oxidation, as well as HO• formation, we show that Cu reacts preferentially with GSH and Cys, halting AscH oxidation and also HO• release. This could be explained by the formation of Cu-thiolate clusters with both GSH and, as we first demonstrate here, Cys. Moreover, we observed a remarkable acceleration of Cu-catalyzed GSH oxidation in the presence of Cys. We provide evidence that both thiol-disulfide exchange and the generated H2O2 contribute to this effect. Based on these findings, we speculate that Cu-induced oxidative stress may be mainly driven by GSH depletion and/or protein disulfide formation rather than by HO• and envision a synergistic effect of Cys on Cu toxicity.

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 x-ray absorption spectroscopy model of solvation about sulfur in aqueous L-cysteine.

The environment of sulfur in dissolved aqueous L-cysteine has been examined using K-edge x-ray absorption spectroscopy (XAS), extended continuum multiple scattering (ECMS) theory, and density functional theory (DFT). For the first time, bound-state and continuum transitions representing the entire XAS spectrum of L-cysteine sulfur are accurately reproduced by theory. Sulfur K-edge absorption features at 2473.3 eV and 2474.2 eV represent transitions to LUMOs that are mixtures of S-C and S-H σ∗ orbitals significantly delocalized over the entire L-cysteine molecule. Continuum features at 2479, 2489, and 2530 eV were successfully reproduced using extended continuum theory. The full L-cysteine sulfur K-edge XAS spectrum could not be reproduced without addition of a water-sulfur hydrogen bond. Density functional theory analysis shows that although the Cys(H)S⋯H-OH hydrogen bond is weak (∼2 kcal) the atomic charge on sulfur is significantly affected by this water. MXAN analysis of hydrogen-bonding structures for L-cysteine and water yielded a best fit model featuring a tandem of two water molecules, 2.9 Å and 5.8 Å from sulfur. The model included a S(cys)⋯H-O(w1)H hydrogen-bond of 2.19 Å and of 2.16 Å for H(2)O(w1)⋯H-O(w2)H. One hydrogen-bonding water-sulfur interaction alone was insufficient to fully describe the continuum XAS spectrum. However, density functional theoretical results are convincing that the water-sulfur interaction is weak and should be only transient in water solution. The durable water-sulfur hydrogen bond in aqueous L-cysteine reported here therefore represents a break with theoretical studies indicating its absence. Reconciling the apparent disparity between theory and result remains the continuing challenge.

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.

Is styrene competitive for dopamine receptor binding?

The potential role of styrene oxide in altering the dopaminergic pathway in the ear is investigated by means of molecular docking and molecular dynamics simulations. We estimate the binding affinity of both styrene oxide and dopamine to the dopaminergic receptor DrD2 by computing the free-energy difference, ∆G, between the configuration where the ligand is bound to the receptor and the situation in which it is "infinitely" far away from it. The results show that the styrene oxide has a somewhat lower affinity for binding with respect to dopamine, which, however, may not be enough to prevent exogenous high concentration styrene oxide to compete with endogenous dopamine for DrD2 binding.

A Thermodynamic Study on the Interaction between RH-23 Peptide and DMPC-Based Biomembrane Models.

Investigation of the interaction between drugs and biomembrane models, as a strategy to study and eventually improve drug/substrate interactions, is a crucial factor in preliminary screening. Synthesized peptides represent a source of potential anticancer and theragnostic drugs. In this study, we investigated the interaction of a novel synthesized peptide, called RH-23, with a simplified dimyristoylphosphatidylcholine (DMPC) model of the cellular membrane. The interaction of RH-23 with DMPC, organized either in multilamellar vesicles (MLVs) and in Langmuir-Blodgett (LB) monolayers, was assessed using thermodynamic techniques, namely differential scanning calorimetry (DSC) and LB. The calorimetric evaluations showed that RH-23 inserted into MLVs, causing a stabilization of the phospholipid gel phase that increased with the molar fraction of RH-23. Interplay with LB monolayers revealed that RH-23 interacted with DMPC molecules. This work represents the first experimental thermodynamic study on the interaction between RH-23 and a simplified model of the lipid membrane, thus providing a basis for further evaluations of the effect of RH-23 on biological membranes and its therapeutic/diagnostic potential.

Zinc modulates copper coordination mode in prion protein octa-repeat subdomains.

In this work we present and analyse XAS measurements carried out on various portions of Prion-protein tetra-octa-repeat peptides in complexes with Cu(II) ions, both in the presence and in the absence of Zn(II). Because of the ability of the XAS technique to provide detailed local structural information, we are able to demonstrate that Zn acts by directly interacting with the peptide, in this way competing with Cu for binding with histidine. This finding suggests that metal binding competition can be important in the more general context of metal homeostasis.

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.

An XAS study of the sulfur environment in human neuromelanin and its synthetic analogs.

Neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in Parkinson's disease. It has been shown to play either a protective or a toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. Understanding its structure and synthesis mechanisms is mandatory to clarify the reason for this remarkable dual behavior. In the present study, X-ray absorption spectroscopy is employed to investigate the sulfur binding mode in natural human neuromelanin, synthetic neuromelanins, and in certain structurally known model compounds, namely cysteine and decarboxytrichochrome C. Based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine- and trichochrome-like sulfur coordination modes is recognized, and the relative abundance of these two types of structural arrangement is determined. Data on the amount of cysteine- and trichochrome-like sulfur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. The interest of the method described here lies in the fact that it allows the identification of different sulfur coordination environments in a physically nondestructive way.

Identifying the structure of the active sites of human recombinant prolidase.

In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (prolidase deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.