Molecular Origins of the Mendelian Rare Diseases Reviewed by Orpha.net: A Structural Bioinformatics Investigation.

The study of rare diseases is important not only for the individuals affected but also for the advancement of medical knowledge and a deeper understanding of human biology and genetics. The wide repertoire of structural information now available from reliable and accurate prediction methods provides the opportunity to investigate the molecular origins of most of the rare diseases reviewed in the Orpha.net database. Thus, it has been possible to analyze the topology of the pathogenic missense variants found in the 2515 proteins involved in Mendelian rare diseases (MRDs), which form the database for our structural bioinformatics study. The amino acid substitutions responsible for MRDs showed different mutation site distributions at different three-dimensional protein depths. We then highlighted the depth-dependent effects of pathogenic variants for the 20,061 pathogenic variants that are present in our database. The results of this structural bioinformatics investigation are relevant, as they provide additional clues to mitigate the damage caused by MRD.

A possible strategy to fight COVID-19: Interfering with spike glycoprotein trimerization.

The recent release of COVID-19 spike glycoprotein allows detailed analysis of the structural features that are required for stabilizing the infective form of its quaternary assembly. Trying to disassemble the trimeric structure of COVID-19 spike glycoprotein, we analyzed single protomer surfaces searching for concave moieties that are located at the three protomer-protomer interfaces. The presence of some druggable pockets at these interfaces suggested that some of the available drugs in Drug Bank could destabilize the quaternary spike glycoprotein formation by binding to these pockets, therefore interfering with COVID-19 life cycle. The approach we propose here can be an additional strategy to fight against the deadly virus. Ligands of COVID-19 spike glycoprotein that we have predicted in the present computational investigation, might be the basis for new experimental studies in vitro and in vivo.

Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra.

The effect of extrinsic paramagnetic probes on NMR relaxation rates for surface mapping of proteins and other biopolymers is a widely investigated and powerful NMR technique. Here we describe a new application of those probes. It relies on the setting of the relaxation delay to generate magnetization equilibrium and off-equilibrium conditions, in order to tailor the extent of steady state signal recovery with and without the water-soluble nitroxide Tempol. With this approach it is possible to identify signals whose relaxation is affected by exchange processes and, from the relative assignments, to map the protein residues involved in association or conformational interconversion processes on a micro-to-millisecond time scale. This finding is confirmed by the comparison with the results obtained from relaxation dispersion measurements. This simple and convenient method allows preliminary inspection to highlight regions where structural or chemical exchange events are operative, in order to focus on quantitative subsequent determinations by transverse relaxation dispersion experiments or analogous NMR relaxation studies, and/or to gain insights into the predictions of calculations.

Correction: Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra.

Correction for 'Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra' by Yamanappa Hunashal et al., Phys. Chem. Chem. Phys., 2020, 22, 6247-6259, DOI: .

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa.

SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria in vitro and in vivo. Insights into its killing mechanism could elucidate correlations with selectivity. SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of P. aeruginosa and K. pneumoniae. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in K. pneumoniae and P. aeruginosa cells. NMR analysis and CD spectra in the presence of sodium dodecyl sulfate micelles showed a transition from an unstructured state to a stable α-helix, driving the peptide to arrange itself on the surface of micelles. SET-M33 kills Gram-negative bacteria after an initial interaction with bacterial LPS. The molecule becomes then embedded in the outer membrane surface, thereby impairing cell function. This activity of SET-M33, in contrast to other similar antimicrobial peptides such as colistin, does not generate resistant mutants after 24h of exposure, non-specific interactions or toxicity against eukaryotic cell membranes, suggesting that SET-M33 is a promising new option for the treatment of Gram-negative antibiotic-resistant infections.

Hot spot mapping of protein surfaces with TEMPOL: Bovine pancreatic RNase A as a model system.

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in the development of reliable surface druggability predictors.

Homogentisic acid induces aggregation and fibrillation of amyloidogenic proteins.

BACKGROUND: Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis. METHODS: Gel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aß(1-42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA. RESULTS: We found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA. CONCLUSIONS: HGA might be an important amyloid co- component playing significant roles in AKU amyloidosis. GENERAL SIGNIFICANCE: Our results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking.

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs.

Cold Denaturation Unveiled: Molecular Mechanism of the Asymmetric Unfolding of Yeast Frataxin.

What is the mechanism that determines the denaturation of proteins at low temperatures, which is, by now, recognized as a fundamental property of all proteins? We present experimental evidence that clarifies the role of specific interactions that favor the entrance of water into the hydrophobic core, a mechanism originally proposed by Privalov but never proved experimentally. By using a combination of molecular dynamics simulation, molecular biology, and biophysics, we identified a cluster of negatively charged residues that represents a preferential gate for the entrance of water molecules into the core. Even single-residue mutations in this cluster, from acidic to neutral residues, affect cold denaturation much more than heat denaturation, suppressing cold denaturation at temperatures above zero degrees. The molecular mechanism of the cold denaturation of yeast frataxin is intrinsically different from that of heat denaturation.

Atom depth analysis delineates mechanisms of protein intermolecular interactions.

The systematic analysis of amino acid distribution, performed inside a large set of resolved protein structures, sheds light on possible mechanisms driving non random protein-protein approaches. Protein Data Bank entries have been selected using as filters a series of restrictions ensuring that the shape of protein surface is not modified by interactions with large or small ligands. 3D atom depth has been evaluated for all the atoms of the 2,410 selected structures. The amino acid relative population in each of the structural layers formed by grouping atoms on the basis of their calculated depths, has been evaluated. We have identified seven structural layers, the inner ones reproducing the core of proteins and the outer one incorporating their most protruding moieties. Quantitative analysis of amino acid contents of structural layers identified, as expected, different behaviors. Atoms of Q, R, K, N, D residues are increasingly more abundant in going from core to surfaces. An opposite trend is observed for V, I, L, A, C, and G. An intermediate behavior is exhibited by P, S, T, M, W, H, F and Y. The outer structural layer hosts predominantly E and K residues whose charged moieties, protruding from outer regions of the protein surface, reorient free from steric hindrances, determining specific electrodynamics maps. This feature may represent a protein signature for long distance effects, driving the formation of encounter complexes and the eventual short distance approaches that are required for protein-protein functional interactions.

Probing the protein space for extending the detection of weak homology folds.

Redundancy of prediction methods has been used to explore the occurrence of weak homology protein motifs. A hybrid template-based algorithm has been implemented to predict different layers of protein structure by detecting domain building sub-structures, which share low sequence identity. Physicochemical determinants, secondary structure profiles, and multiple alignments have been analyzed to generate a broad set of structural sub-domains. Then, intensive computing procedures generated all the various tridimensional folds on the basis of secondary structure predictions, fragment assembly and detection of structural homologs. The proposed algorithm not only identifies common protein sub-structures, but also detects higher order architectures such as domain superfamilies/superfolds by linking backbone trajectories of supersecondary structures. Applying rigid transformation protocols, population of the detected domain building models with an average root mean square deviation from native structures of 2.3Å and an average template modeling score from native structures of 0.43 has been obtained. The fold detection algorithm here proposed yields more accurate results than previously proposed methods, predicting structural homology also for proteins sharing less than 20% sequence identity. Our tools are freely available at http://www.acbrc.org/tools.html.

Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions.

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

Structural Bioinformatic Survey of Protein-Small Molecule Interfaces Delineates the Role of Glycine in Surface Pocket Formation.

With a structural bioinformatic approach, we have explored amino acid compositions at PISA defined interfaces between small molecules and proteins that are contained in an optimized subset of 11,351 PDB files. The use of a series of restrictions, to prevent redundancy and biases from interactions between amino acids with charged side chains and ions, yielded a final data set of 45,230 protein-small molecule interfaces. We have compared occurrences of natural amino acids in surface exposed regions and binding sites for all the proteins of our data set. From our structural bioinformatic survey, the most relevant signal arose from the unexpected Gly abundance at enzyme catalytic sites. This finding suggested that Gly must have a fundamental role in stabilizing concave protein surface moieties. Subsequently, we have tried to predict the effect of in silico Gly mutations in hen egg white lysozyme to optimize those conditions that can reshape the protein surface with the appearance of new pockets. Replacing amino acids having bulky side chains with Gly in specific protein regions seems a feasible way for designing proteins with additional surface pockets, which can alter protein surface dynamics, therefore, representing controllable switches for protein activity.

A Bioinformatics Approach to Investigate Structural and Non-Structural Proteins in Human Coronaviruses.

Recent studies confirmed that people unexposed to SARS-CoV-2 have preexisting reactivity, probably due to previous exposure to widely circulating common cold coronaviruses. Such preexistent reactivity against SARS-CoV-2 comes from memory T cells that can specifically recognize a SARS-CoV-2 epitope of structural and non-structural proteins and the homologous epitopes from common cold coronaviruses. Therefore, it is important to understand the SARS-CoV-2 cross-reactivity by investigating these protein sequence similarities with those of different circulating coronaviruses. In addition, the emerging SARS-CoV-2 variants lead to an intense interest in whether mutations in proteins (especially in the spike) could potentially compromise vaccine effectiveness. Since it is not clear that the differences in clinical outcomes are caused by common cold coronaviruses, a deeper investigation on cross-reactive T-cell immunity to SARS-CoV-2 is crucial to examine the differential COVID-19 symptoms and vaccine performance. Therefore, the present study can be a starting point for further research on cross-reactive T cell recognition between circulating common cold coronaviruses and SARS-CoV-2, including the most recent variants Delta and Omicron. In the end, a deep learning approach, based on Siamese networks, is proposed to accurately and efficiently calculate a BLAST-like similarity score between protein sequences.

Hydration studies on the archaeal protein Sso7d using NMR measurements and MD simulations.

BACKGROUND: How proteins approach surrounding molecules is fundamental to our understanding of the specific interactions that occur at the surface of proteins. The enhanced surface accessibility of small molecules such as organic solvents and paramagnetic probes to protein binding sites has been observed; however, the molecular basis of this finding has not been fully established. Recently, it has been suggested that hydration dynamics play a predominant role in controlling the distribution of hot spots on surface of proteins. RESULTS: In the present study, the hydration of the archaeal multifunctional protein Sso7d from Solfolobus solfataricus was investigated using a combination of computational and experimental data derived from molecular dynamics simulations and ePHOGSY NMR spectroscopy. CONCLUSIONS: We obtained a convergent protein hydration landscape that indicated how the shape and stability of the Sso7d hydration shell could modulate the function of the protein. The DNA binding domain overlaps with the protein region involved in chaperon activity and this domain is hydrated only in a very small central region. This localized hydration seems to favor intermolecular approaches from a large variety of ligands. Conversely, high water density was found in surface regions of the protein where the ATP binding site is located, suggesting that surface water molecules play a role in protecting the protein from unspecific interactions.

Structural bioinformatics survey on disease-inducing missense mutations.

Understanding the molecular mechanisms that correlate pathologies with missense mutations is of critical importance for disease risk estimations and for devising personalized therapies. Thus, we have performed a bioinformatic survey of ClinVar, a database of human genomic variations, to find signals that can account for missense mutation pathogenicity. Arginine resulted as the most frequently replaced amino acid both in benign and pathogenic mutations. By adding the structural dimension to this investigation to increase its resolution, we found that arginine mutations occurring at the protein-DNA interface increase pathogenicity 6.5 times with respect to benign variants. Glycine is the second amino acid among all the pathological missense mutations. Necessarily replaced by larger amino acids, glycine substitutions perturb the structural stability of proteins and, therefore, their functions, being mostly located in buried protein moieties. Arginine and glycine appear as representative of missense mutations causing respective changes in interaction processes and protein structural features, the two main molecular mechanisms of genome-induced pathologies.

Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL.

The surface accessibility of macromolecules plays a key role in modulating molecular recognition events. RNA is a complex and dynamic molecule involved in many aspects of gene expression. However, there are few experimental methods available to measure the accessible surface of RNA. Here, we investigate the accessible surface of RNA using NMR and the small paramagnetic molecule TEMPOL. We investigated two RNAs with known structures, one that is extremely stable and one that is dynamic. For helical regions, the TEMPOL probing data correlate well with the predicted RNA surface, and the method is able to distinguish subtle variations in atom depths, such as the relative accessibility of pyrimidine versus purine aromatic carbon atoms. Dynamic motions are also detected by TEMPOL probing, and the method accurately reports a previously characterized pH-dependent conformational transition involving formation of a protonated C-A pair and base flipping. Some loop regions are observed to exhibit anomalously high accessibility, reflective of motions that are not evident within the ensemble of NMR structures. We conclude that TEMPOL probing can provide valuable insights into the surface accessibility and dynamics of RNA, and can also be used as an independent means of validating RNA structure and dynamics in solution.

Glycine-induced formation and druggability score prediction of protein surface pockets.

Nowadays, it is well established that most of the human diseases which are not related to pathogen infections have their origin from DNA disorders. Thus, DNA mutations, waiting for the availability of CRISPR-like remedies, will propagate into proteomics, offering the possibility to select natural or synthetic molecules to fight against the effects of malfunctioning proteins. Drug discovery, indeed, is a flourishing field of biotechnological research to improve human health, even though the development of a new drug is increasingly more expensive in spite of the massive use of informatics in Medicinal Chemistry. CRISPR technology adds new alternatives to cure diseases by removing DNA defects responsible of genome-related pathologies. In principle, the same technology, however, could also be exploited to induce protein mutations whose effects are controlled by the presence of suitable ligands. In this paper, a new idea is proposed for the realization of mutated proteins, on the surface of which more spacious transient pockets are formed and, therefore, are more suitable for hosting drugs. In particular, new allosteric sites are obtained by replacing amino-acids with bulky side chains with glycine, Gly, the smallest natural amino-acid. We also present a machine learning approach to evaluate the druggability score of new (or enlarged) pockets. Preliminary experimental results are very promising, showing that 10% of the sites created by the Gly-pipe software are druggable.

Structural investigation of Rett-inducing MeCP2 mutations.

X-ray structure of methyl-CpG binding domain (MBD) of MeCP2, an intrinsically disordered protein (IDP) involved in Rett syndrome, offers a rational basis for defining the spatial distribution for most of the sites where mutations responsible of Rett syndrome, RTT, occur. We have ascribed pathogenicity for mutations of amino acids bearing positively charged side chains, all located at the protein-DNA interface, as positive charge removal cause reduction of the MeCP2-DNA adduct lifetime. Pathogenicity of the frequent proline replacements, outside the DNA contact moiety of MBD, can be attributed to the role of this amino acid for maintaining both unfolded states for unbound MeCP2 and, at the same time, to favor some higher conformational order for stabilizing structural determinants required by protein activity. These hypotheses can be extended to transcription repressor domain, TRD, the other MeCP2-DNA interaction site and, in general, to all the IDP that interact with nucleic acids.

Peptides and small molecules blocking the CXCR4/CXCL12 axis overcome bone marrow­induced chemoresistance in acute leukemias.

Notable advances in treatment have been made and increases in the cure rates of pediatric leukemia have been achieved. However, the majority of children with relapsed disease are not expected to survive, with chemotherapy resistance acting as the principal cause of treatment failure. Interaction between leukemic cells and the bone marrow microenvironment is the primary cause of relapse. It was identified that a multi­protein membrane complex, formed by potassium voltage­gated channel subfamily H member 2 (hERG1) channels, the ß1 integrin subunit and the stromal cell­derived factor 12 (CXCL12) receptor, C­X­C chemokine receptor type 4 (CXCR4), exerts a role in mesenchymal stromal cell (MSC)­mediated chemoresistance in pediatric leukemias. hERG1 blockade was able to overcome chemoresistance in vitro and in vivo. As an alternative strategy to overcome chemoresistance, the present study evaluated the effects of novel tools targeting the CXCR4/CXCL12 axis. The analysis of CXCL12 structural dynamics was used for the selection of a peptide (4­1­17) and a small molecule (8673), which interact with a transient hot spot, identified by a dynamic drug design approach. The present findings indicated that peptide 4­1­17 and small molecule 8673 inhibited leukemia cell proliferation and induced a pro­apoptotic effect, which was not reduced by the presence of MSCs. The combined treatment with 4­1­17 and 8673 had a stronger pro­apoptotic effect, particularly on cells cultured on MSCs in normoxic and hypoxic conditions, and was able to overcome MSC­induced resistance to cytarabine. Overall, the targeting of CXCL12 and the ensuing inhibition of the CXCR4/CXCL12 axis may be proposed as an alternative strategy to overcome chemoresistance in leukemia.