95 results on '"Vito Calderone"'
Search Results
2. Gold-Based Metal Drugs as Inhibitors of Coronavirus Proteins: The Inhibition of SARS-CoV-2 Main Protease by Auranofin and Its Analogs
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Lara Massai, Deborah Grifagni, Alessia De Santis, Andrea Geri, Francesca Cantini, Vito Calderone, Lucia Banci, and Luigi Messori
- Subjects
COVID-19 ,nsp5 ,Mpro ,SARS-CoV-2 ,Auranofin ,gold compounds ,Microbiology ,QR1-502 - Abstract
Gold compounds have a long tradition in medicine and offer many opportunities for new therapeutic applications. Herein, we evaluated the lead compound Auranofin and five related gold(I) complexes as possible inhibitors of SARS-CoV-2 Main Protease (SARS-CoV-2 Mpro), a validated drug target for the COVID-19 disease. The investigational panel of gold compounds included Auranofin; three halido analogues, i.e., Au(PEt3)Cl, Au(PEt3)Br, and Au(PEt3)I; and two gold carbene complexes, i.e., Au(NHC)Cl and [Au(NHC)2]PF6. Notably, all these gold compounds, with the only exception of [Au(NHC)2]PF6, turned out to be potent inhibitors of the catalytic activity of SARS-CoV-2 Mpro: the measured Ki values were in the range 2.1–0.4 μM. The reactions of the various gold compounds with SARS-CoV-2 Mpro were subsequently investigated through electrospray ionization (ESI) mass spectrometry (MS) upon a careful optimization of the experimental conditions; the ESI MS spectra provided clear evidence for the formation of tight metallodrug-protein adducts and for the coordination of well defined gold-containing fragments to the SARS-CoV-2 Mpro, again with the only exception of [Au(NHC)2]PF6, The metal-protein stoichiometry was unambiguously determined for the resulting species. The crystal structures of the metallodrug- Mpro adducts were solved in the case of Au(PEt3)Br and Au(NHC)Cl. These crystal structures show that gold coordination occurs at the level of catalytic Cys 145 in the case of Au(NHC)Cl and at the level of both Cys 145 and Cys 156 for Au(PEt3)Br. Tight coordination of gold atoms to functionally relevant cysteine residues is believed to represent the true molecular basis of strong enzyme inhibition.
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- 2022
- Full Text
- View/download PDF
3. Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins
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Francesca Camponeschi, Simone Ciofi-Baffoni, Vito Calderone, and Lucia Banci
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iron–sulfur cluster ,mitochondrial proteins ,multiple mitochondrial dysfunction syndromes ,rare diseases ,Microbiology ,QR1-502 - Abstract
The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron–sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation. The synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. The present review provides a structural and molecular overview of the rare diseases associated with the genes encoding for the accessory proteins of the ISC machinery (i.e., GLRX5, ISCA1, ISCA2, IBA57, FDX2, BOLA3, IND1 and NFU1) involved in the assembly and insertion of [4Fe-4S] clusters in mitochondrial proteins. The disease-related missense mutations were mapped on the 3D structures of these accessory proteins or of their protein complexes, and the possible impact that these mutations have on their specific activity/function in the frame of the mitochondrial [4Fe-4S] protein biogenesis is described.
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- 2022
- Full Text
- View/download PDF
4. Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii
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Caterina Ciani, Anna Pérez-Ràfols, Isabelle Bonomo, Mariachiara Micaelli, Alfonso Esposito, Chiara Zucal, Romina Belli, Vito Giuseppe D’Agostino, Irene Bianconi, Vito Calderone, Linda Cerofolini, Orietta Massidda, Michael Bernard Whalen, Marco Fragai, and Alessandro Provenzani
- Subjects
Acinetobacter baumannii ,RNA recognition motif ,ELAVL1 ,Microbiology ,QR1-502 - Abstract
Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical β1α1β2β3α2β4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.
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- 2022
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5. Effect of Non-Lethal Selection on Spontaneous Revertants of Frameshift Mutations: The Escherichia coli hisF Case
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Sara Del Duca, Anna Maria Puglia, Vito Calderone, Marco Bazzicalupo, and Renato Fani
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directed-evolution experiment ,reverse mutation ,protein structure ,Biology (General) ,QH301-705.5 - Abstract
Microorganisms possess the potential to adapt to fluctuations in environmental parameters, and their evolution is driven by the continuous generation of mutations. The reversion of auxotrophic mutations has been widely studied; however, little is known about the reversion of frameshift mutations resulting in amino acid auxotrophy and on the structure and functioning of the protein encoded by the revertant mutated gene. The aims of this work were to analyze the appearance of reverse mutations over time and under different selective pressures and to investigate revertant enzymes’ three-dimensional structures and their correlation with a different growth ability. Escherichia coli FB182 strain, carrying the hisF892 single nucleotide deletion resulting in histidine auxotrophy, was subjected to different selective pressures, and revertant mutants were isolated and characterized. The obtained results allowed us to identify different indels of different lengths located in different positions in the hisF gene, and relations with the incubation time and the selective pressure applied were observed. Moreover, the structure of the different mutant proteins was consistent with the respective revertant ability to grow in absence of histidine, highlighting a correlation between the mutations and the catalytic activity of the mutated HisF enzyme.
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- 2022
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6. On the complementarity of X-ray and NMR data
- Author
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Antonio Schirò, Azzurra Carlon, Giacomo Parigi, Garib Murshudov, Vito Calderone, Enrico Ravera, and Claudio Luchinat
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Structure refinement ,Integrated structural biology ,RDC ,X-ray ,REFMAC ,Biology (General) ,QH301-705.5 - Abstract
X-ray crystallography and NMR contain complementary information for the structural characterization of biological macromolecules. X-ray diffraction is primarily sensitive to the overall shape of the molecule, whereas NMR is mostly sensitive to the atomic detail. Their combination can therefore provide a stronger justification for the resulting structure. For their combination we have recently proposed REFMAC-NMR, which relies on primary data from both techniques for joint refinement. This possibility raises the compelling question of how far the complementarity can be extended. In this paper, we describe an integrative approach to the refinement with NMR data of four X-ray structures of hen-egg-white lysozyme, solved at atomic resolution in four different crystal forms, and we demonstrate that the outcome critically depends on the crystal form itself, reflecting the sensitivity of NMR to fine details.
- Published
- 2020
- Full Text
- View/download PDF
7. On the Mechanism of Bioinspired Formation of Inorganic Oxides: Structural Evidence of the Electrostatic Nature of the Interaction between a Mononuclear Inorganic Precursor and Lysozyme
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Lucia Gigli, Enrico Ravera, Vito Calderone, and Claudio Luchinat
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lysozyme ,titanium ,biomineralization ,inorganic oxides ,Microbiology ,QR1-502 - Abstract
Nature has evolved several molecular machineries to promote the formation at physiological conditions of inorganic materials, which would otherwise be formed in extreme conditions. The molecular determinants of this process have been established over the last decade, identifying a strong role of electrostatics in the first steps of the precipitation. However, no conclusive, structure-based evidence has been provided so far. In this manuscript, we test the binding of lysozyme with silica and titania potential precursors. In contrast with the absence of structural information about the interaction with the silica precursor, we observe the interaction with a mononuclear titanium(IV) species, which is found to occur in a region rich of positive charges.
- Published
- 2020
- Full Text
- View/download PDF
8. Integrative Approaches in Structural Biology: A More Complete Picture from the Combination of Individual Techniques
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Linda Cerofolini, Marco Fragai, Enrico Ravera, Christoph A. Diebolder, Ludovic Renault, and Vito Calderone
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integrative structural biology ,X-ray crystallography ,solution NMR ,SSNMR ,SAXS ,cryo-EM ,Microbiology ,QR1-502 - Abstract
With the recent technological and computational advancements, structural biology has begun to tackle more and more difficult questions, including complex biochemical pathways and transient interactions among macromolecules. This has demonstrated that, to approach the complexity of biology, one single technique is largely insufficient and unable to yield thorough answers, whereas integrated approaches have been more and more adopted with successful results. Traditional structural techniques (X-ray crystallography and Nuclear Magnetic Resonance (NMR)) and the emerging ones (cryo-electron microscopy (cryo-EM), Small Angle X-ray Scattering (SAXS)), together with molecular modeling, have pros and cons which very nicely complement one another. In this review, three examples of synergistic approaches chosen from our previous research will be revisited. The first shows how the joint use of both solution and solid-state NMR (SSNMR), X-ray crystallography, and cryo-EM is crucial to elucidate the structure of polyethylene glycol (PEG)ylated asparaginase, which would not be obtainable through any of the techniques taken alone. The second deals with the integrated use of NMR, X-ray crystallography, and SAXS in order to elucidate the catalytic mechanism of an enzyme that is based on the flexibility of the enzyme itself. The third one shows how it is possible to put together experimental data from X-ray crystallography and NMR restraints in order to refine a protein model in order to obtain a structure which simultaneously satisfies both experimental datasets and is therefore closer to the ‘real structure’.
- Published
- 2019
- Full Text
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9. Direct Expression of Fluorinated Proteins in Human Cells for
- Author
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Lan B T, Pham, Azzurra, Costantino, Letizia, Barbieri, Vito, Calderone, Enrico, Luchinat, and Lucia, Banci
- Abstract
In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments.
- Published
- 2023
10. SARS-CoV-2 M
- Author
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Deborah, Grifagni, Vito, Calderone, Stefano, Giuntini, Francesca, Cantini, Marco, Fragai, and Lucia, Banci
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Zinc ,Binding Sites ,Protein Conformation ,SARS-CoV-2 ,COVID-19 ,Humans ,Crystallography, X-Ray ,Coronavirus 3C Proteases - Abstract
Structural data on the SARS-CoV-2 main protease in complex with a zinc-containing organic inhibitor are already present in the literature and gave hints on the presence of a zinc binding site involving the catalytically relevant cysteine and histidine residues. In this paper, the structural basis of ionic zinc binding to the SARS-CoV-2 main protease has been elucidated by X-ray crystallography. The zinc binding affinity and its ability to inhibit the SARS-CoV-2 main protease have been investigated. These findings provide solid ground for the design of potent and selective metal-conjugated inhibitors of the SARS-CoV-2 main protease.
- Published
- 2021
11. A high‐resolution view of the coordination environment in a paramagnetic metalloprotein from its magnetic properties
- Author
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Elizaveta A. Suturina, Vito Calderone, Giacomo Parigi, Claudio Luchinat, Marco Fragai, Lucia Gigli, and Enrico Ravera
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Materials science ,010405 organic chemistry ,Magnetic Phenomena ,Metal ions in aqueous solution ,Resolution (electron density) ,General Medicine ,Nuclear magnetic resonance spectroscopy ,General Chemistry ,Energy minimization ,010402 general chemistry ,01 natural sciences ,Catalysis ,Characterization (materials science) ,0104 chemical sciences ,Metal ,Paramagnetism ,Chemical physics ,visual_art ,Metalloproteins ,visual_art.visual_art_medium ,Diamagnetism ,Condensed Matter::Strongly Correlated Electrons ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Metalloproteins constitute a significant fraction of the proteome of all organisms and their characterization is critical for both basic sciences and biomedical applications. A large portion of metalloproteins bind paramagnetic metal ions, and paramagnetic NMR spectroscopy has been widely used in their structural characterization. However, the signals of nuclei in the immediate vicinity of the metal center are often broadened beyond detection. In this work, we show that it is possible to determine the coordination environment of the paramagnetic metal in the protein at a resolution inaccessible to other techniques. Taking the structure of a diamagnetic analogue as a starting point, a geometry optimization is carried out by fitting the pseudocontact shifts obtained from first principles quantum chemical calculations to the experimental ones.
- Published
- 2021
12. Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics
- Author
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Enrico Ravera, Giacomo Parigi, Azzurra Carlon, Claudio Luchinat, Stefano Giuntini, Marco Fragai, Linda Cerofolini, and Vito Calderone
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Magnetic Resonance Spectroscopy ,Combinatorial chemistry ,Hydrolase ,Computational biology ,PEGylation ,biopharmaceuticals ,protein modifications ,protein structures ,structural biology ,010402 general chemistry ,01 natural sciences ,Catalysis ,Polyethylene Glycols ,Protein structure ,Coated Materials, Biocompatible ,Asparaginase ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Rational design ,General Chemistry ,Integrated approach ,0104 chemical sciences ,3. Good health ,Characterization (materials science) ,Structural biology ,Pegylated asparaginase ,Protein Multimerization ,Peg coating - Abstract
Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.
- Published
- 2019
13. Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins
- Author
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Vito Calderone, Francesca Camponeschi, LUCIA BANCI, and Simone Ciofi Baffoni
- Subjects
Iron-Sulfur Proteins ,Mitochondrial Proteins ,Rare Diseases ,Iron ,Humans ,Molecular Biology ,Biochemistry ,Sulfur ,Mitochondria - Abstract
The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron–sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation. The synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. The present review provides a structural and molecular overview of the rare diseases associated with the genes encoding for the accessory proteins of the ISC machinery (i.e., GLRX5, ISCA1, ISCA2, IBA57, FDX2, BOLA3, IND1 and NFU1) involved in the assembly and insertion of [4Fe-4S] clusters in mitochondrial proteins. The disease-related missense mutations were mapped on the 3D structures of these accessory proteins or of their protein complexes, and the possible impact that these mutations have on their specific activity/function in the frame of the mitochondrial [4Fe-4S] protein biogenesis is described.
- Published
- 2022
14. Non-crystallographic symmetry in proteins: Jahn–Teller-like and Butterfly-like effects?
- Author
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Linda Cerofolini, Anjos L. Macedo, Marco Fragai, Enrico Ravera, Claudio Luchinat, José Malanho Silva, Stefano Giuntini, Vito Calderone, and Carlos F. G. C. Geraldes
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Models, Molecular ,Protein Conformation ,Jahn–Teller effect ,media_common.quotation_subject ,Crystallography, X-Ray ,010402 general chemistry ,Carbonic Anhydrase II ,01 natural sciences ,Biochemistry ,Asymmetry ,Inorganic Chemistry ,Crystal ,Nickel ,Humans ,Molecule ,media_common ,Crystallographic point group ,Binding Sites ,biology ,010405 organic chemistry ,Chemistry ,Active site ,Symmetry (physics) ,0104 chemical sciences ,Crystallography ,X-ray crystallography ,biology.protein - Abstract
Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.
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- 2018
15. A fragment-based approach identifies an allosteric pocket that impacts malate dehydrogenase activity
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Alexander Dömling, Atilio Reyes Romero, Michał Taube, Vito Calderone, Maciej Kozak, Matteo Gentili, Tad A. Holak, Michael Sattler, Grzegorz M Popowicz, Matthew Groves, Serjey Lunev, Jacek Plewka, Drug Design, and Medicinal Chemistry and Bioanalysis (MCB)
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Models, Molecular ,QH301-705.5 ,Allosteric regulation ,Plasmodium falciparum ,Protozoan Proteins ,Biophysics ,Medicine (miscellaneous) ,Sequence (biology) ,Malate dehydrogenase ,General Biochemistry, Genetics and Molecular Biology ,Article ,Catalytic Domain ,Binding site ,Biology (General) ,chemistry.chemical_classification ,Binding Sites ,biology ,Drug discovery ,Active site ,Small molecule ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,General Agricultural and Biological Sciences - Abstract
Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site., Romero et al. perform NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface of malate dehydrogenase (MDH). Their study indicates an allosteric mechanism impacting enzymatic activity, paving the way for development of more selective molecules and a starting point for the future development of specific MDH inhibitors.
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- 2021
16. SARS-CoV-2 Mproinhibition by a zinc ion: structural features and hints for drug design
- Author
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Marco Fragai, Stefano Giuntini, Deborah Grifagni, Francesca Cantini, Vito Calderone, and Lucia Banci
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0301 basic medicine ,Drug ,Stereochemistry ,media_common.quotation_subject ,medicine.medical_treatment ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,chemistry.chemical_element ,Zinc ,010402 general chemistry ,01 natural sciences ,Catalysis ,03 medical and health sciences ,Protein structure ,Binding Sites ,COVID-19 ,Coronavirus 3C Proteases ,Crystallography, X-Ray ,Humans ,Protein Conformation ,SARS-CoV-2 ,Materials Chemistry ,medicine ,Binding site ,skin and connective tissue diseases ,Histidine ,media_common ,Protease ,Chemistry ,fungi ,Metals and Alloys ,General Chemistry ,0104 chemical sciences ,3. Good health ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,body regions ,030104 developmental biology ,Ceramics and Composites ,Cysteine - Abstract
Structural data on the SARS-CoV-2 main protease in complex with a zinc-containing organic inhibitor are already present in the literature and gave hints on the presence of a zinc binding site involving the catalytically relevant cysteine and histidine residues. In this paper, the structural basis of ionic zinc binding to the SARS-CoV-2 main protease has been elucidated by X-ray crystallography. The zinc binding affinity and its ability to inhibit the SARS-CoV-2 main protease have been investigated. These findings provide solid ground for the design of potent and selective metal-conjugated inhibitors of the SARS-CoV-2 main protease.
- Published
- 2021
17. Supplementary material to 'Revisiting paramagnetic relaxation enhancements in slowly rotating systems: how long is the long range?'
- Author
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Giovanni Bellomo, Enrico Ravera, Vito Calderone, Mauro Botta, Marco Fragai, Giacomo Parigi, and Claudio Luchinat
- Published
- 2020
18. On the Mechanism of Bioinspired Formation of Inorganic Oxides: Structural Evidence of the Electrostatic Nature of the Interaction between a Mononuclear Inorganic Precursor and Lysozyme
- Author
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Vito Calderone, Enrico Ravera, Lucia Gigli, and Claudio Luchinat
- Subjects
Static Electricity ,lcsh:QR1-502 ,chemistry.chemical_element ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,lcsh:Microbiology ,Article ,chemistry.chemical_compound ,Molecular Biology ,lysozyme ,Titanium ,Precipitation (chemistry) ,Oxides ,021001 nanoscience & nanotechnology ,Electrostatics ,biomineralization ,0104 chemical sciences ,chemistry ,Chemical engineering ,Mechanism (philosophy) ,Inorganic Chemicals ,titanium ,inorganic oxides ,Inorganic materials ,Muramidase ,Lysozyme ,0210 nano-technology ,Biomineralization - Abstract
Nature has evolved several molecular machineries to promote the formation at physiological conditions of inorganic materials, which would otherwise be formed in extreme conditions. The molecular determinants of this process have been established over the last decade, identifying a strong role of electrostatics in the first steps of the precipitation. However, no conclusive, structure-based evidence has been provided so far. In this manuscript, we test the binding of lysozyme with silica and titania potential precursors. In contrast with the absence of structural information about the interaction with the silica precursor, we observe the interaction with a mononuclear titanium(IV) species, which is found to occur in a region rich of positive charges.
- Published
- 2020
19. On the complementarity of X-ray and NMR data
- Author
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Giacomo Parigi, Vito Calderone, Antonio Schirò, Enrico Ravera, Azzurra Carlon, Claudio Luchinat, and Garib N. Murshudov
- Subjects
Diffraction ,Materials science ,Complementarity (molecular biology) ,010402 general chemistry ,Macromolecule ,RDC ,01 natural sciences ,Article ,Crystal ,X-ray ,03 medical and health sciences ,Structural Biology ,REFMAC ,Molecule ,Atomic resolution ,Structure refinement ,Integrated structural biology ,lcsh:QH301-705.5 ,030304 developmental biology ,ComputingMethodologies_COMPUTERGRAPHICS ,0303 health sciences ,Crystallography ,Nmr data ,0104 chemical sciences ,3. Good health ,Characterization (materials science) ,lcsh:Biology (General) ,Chemical physics - Abstract
Graphical abstract, Highlights • X-ray diffraction and NMR spectroscopy are complementary sources of information. • Even at atomic resolution, NMR data improves the accuracy of X-ray structures. • Real local discrepancies between solution and crystals are highlighted. • For HEWL, a better agreement is found for the crystal forms with looser packing., X-ray crystallography and NMR contain complementary information for the structural characterization of biological macromolecules. X-ray diffraction is primarily sensitive to the overall shape of the molecule, whereas NMR is mostly sensitive to the atomic detail. Their combination can therefore provide a stronger justification for the resulting structure. For their combination we have recently proposed REFMAC-NMR, which relies on primary data from both techniques for joint refinement. This possibility raises the compelling question of how far the complementarity can be extended. In this paper, we describe an integrative approach to the refinement with NMR data of four X-ray structures of hen-egg-white lysozyme, solved at atomic resolution in four different crystal forms, and we demonstrate that the outcome critically depends on the crystal form itself, reflecting the sensitivity of NMR to fine details.
- Published
- 2020
20. When molecular replacement has no trivial solution: The importance of model editing in human S100Z X-ray structure solution
- Author
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Vito Calderone, Claudio Luchinat, and Marco Fragai
- Subjects
0301 basic medicine ,Structure (mathematical logic) ,Crystallographic point group ,Chemistry ,business.industry ,Nanotechnology ,Phase problem ,Type (model theory) ,Inorganic Chemistry ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Software ,Trivial solution ,Materials Chemistry ,Molecular replacement ,Physical and Theoretical Chemistry ,High order ,business ,Algorithm ,030215 immunology - Abstract
Molecular replacement (MR) is normally a successful technique to solve the phase problem in protein X-ray crystallography. Its use is more and more common, taking advantage of the ever increasing number of structures deposited in the PDB; in MR a structure homologous to the target one is used as a template to obtain the phase values needed for computing electron density. There are however situations that may hinder MR success, such as low diffraction data quality, pseudo-translation, twinning, high order non crystallographic symmetry (NCS) within the asymmetric unit and, last but not least, the quality of the model used as the template. Solving the X-ray structure of human apo-S100Z by MR turned out to be a challenge and required trying several approaches and software tools; in an attempt to help researchers who may encounter this type of difficulties, we illustrate the attempted and failed MR approaches and speculate on the plausible reasons for which only certain approaches allowed to overcome the difficulties encountered. More importantly, we stress the crucial importance of a proper model editing for a successful protein structure solution through MR.
- Published
- 2018
21. Lipoyl-Homotaurine Derivative (ADM_12) Reverts Oxaliplatin-Induced Neuropathy and Reduces Cancer Cells Malignancy by Inhibiting Carbonic Anhydrase IX (CAIX)
- Author
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Barbara Richichi, Lorenzo Di Cesare Mannelli, Alexandra Louka, Oscar Francesconi, Cristina Nativi, Marco Fragai, Andrea Angeli, Vito Calderone, Roberta Gualdani, Giuseppina Comito, Paola Chiarugi, Carla Ghelardini, Alessio Nocentini, Paola Gratteri, Francesco Tadini-Buoninsegni, and Claudiu T. Supuran
- Subjects
0301 basic medicine ,Organoplatinum Compounds ,Taurine ,Stereochemistry ,Antineoplastic Agents ,03 medical and health sciences ,chemistry.chemical_compound ,In vivo ,Cell Line, Tumor ,Neoplasms ,Pancreatic cancer ,Drug Discovery ,medicine ,Humans ,Carbonic Anhydrase IX ,Carbonic Anhydrase Inhibitors ,TRPA1 Cation Channel ,Antagonist ,Peripheral Nervous System Diseases ,Drug Synergism ,medicine.disease ,In vitro ,Oxaliplatin ,Pancreatic Neoplasms ,030104 developmental biology ,chemistry ,Homotaurine ,Cell culture ,Cancer cell ,Cancer research ,Molecular Medicine ,medicine.drug - Abstract
Oxaliplatin (OXA) is a valuable and largely used cancer drug which induces a serious and intractable neuropathy. The lipoyl-homotaurine derivative (ADM_12) reverts in vivo OXA-induced neuropathy, and it is an effective antagonist of the nociceptive sensor channel TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells' aggressiveness.
- Published
- 2017
22. Exploration of zinc-binding groups for the design of inhibitors for the oxytocinase subfamily of M1 aminopeptidases
- Author
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Magdalini Kakou, Despoina Koumantou, Marco Fragai, Sofia Tsoukalidou, Vito Calderone, Efstratios Stratikos, Athanasios Papakyriakou, Dionisios Vourloumis, and Ioannis Mavridis
- Subjects
Models, Molecular ,Subfamily ,Protein Conformation ,Clinical Biochemistry ,Pharmaceutical Science ,chemistry.chemical_element ,Zinc ,01 natural sciences ,Biochemistry ,Aminopeptidase ,Aminopeptidases ,Gene Expression Regulation, Enzymologic ,Minor Histocompatibility Antigens ,Structure-Activity Relationship ,Drug Discovery ,Hydrolase ,Molecular Biology ,chemistry.chemical_classification ,Metalloproteinase ,Binding Sites ,biology ,010405 organic chemistry ,Organic Chemistry ,Active site ,Affinities ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Interleukin 1 Receptor Antagonist Protein ,Enzyme ,chemistry ,Drug Design ,biology.protein ,Molecular Medicine ,Protein Binding - Abstract
The oxytocinase subfamily of M1 aminopeptidases consists of three members, ERAP1, ERAP2 and IRAP that play several important biological roles, including key functions in the generation of antigenic peptides that drive human immune responses. They represent emerging targets for pharmacological manipulation of the immune system, albeit lack of selective inhibitors is hampering these efforts. Most of the previously explored small-molecule binders target the active site of the enzymes via strong interactions with the catalytic zinc(II) atom and, while achieving increased potency, they suffer in selectivity. Continuing our earlier efforts on weaker zinc(II) binding groups (ZBG), like the 3,4-diaminobenzoic acid derivatives (DABA), we herein synthesized and biochemically evaluated analogues of nine potentially weak ZBGs, based on differential substitutions of functionalized pyridinone- and pyridinethione-scaffolds, nicotinic-, isonicotinic-, aminobenzoic- and hydrazinobenzoic-acids. Crystallographic analysis of two analogues in complex with a metalloprotease (MMP-12) revealed unexpected binding topologies, consistent with the observed affinities. Our results suggest that the potency of the compounds as inhibitors of ERAP1, ERAP2 and IRAP is primarily driven by the occupation of active-site specificity pockets and their proper orientation within the enzymes.
- Published
- 2019
23. Reviewing the Crystal Structure of S100Z and Other Members of the S100 Family: Implications in Calcium-Regulated Quaternary Structure
- Author
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Vito, Calderone, Marco, Fragai, and Claudio, Luchinat
- Subjects
Models, Molecular ,S100 Proteins ,Animals ,Humans ,Sequence Homology ,Calcium ,Protein Multimerization ,Zebrafish Proteins ,Crystallography, X-Ray ,Protein Structure, Quaternary ,Zebrafish ,Protein Binding - Abstract
This paper takes the cue from the previously solved crystal structure of human apo-S100Z and compares it with that of the calcium-bound S100Z from zebrafish in order to stress, for this particular S100, the significant role of the presence of calcium in promoting supramolecular assemblies with likely biological meaning. This consideration is then expanded through a wider review on analogous situations concerning all other S100s for which there is crystallographic o biochemical evidence of how the presence of calcium promotes the formation of quaternary complexes.The paper also deals with some considerations on the quality of the crystals obtained for the solved members of this family and on the need for experimental phasing for solving some of the structures where the good general sequence homology among the members of the family would have suggested molecular replacement (MR) as the easiest way to solve them.These considerations, along with the PCA analysis carried out on all the known S100s, further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation for several members of this family of proteins.
- Published
- 2019
24. Metal centers in biomolecular solid-state NMR
- Author
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Vito Calderone, Anjos L. Macedo, Linda Cerofolini, Giacomo Parigi, Carlos F. G. C. Geraldes, Enrico Ravera, Marco Fragai, Claudio Luchinat, José Malanho Silva, and Stefano Giuntini
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Materials science ,Protein Conformation ,Iron ,Nanotechnology ,Electronic structure ,Metal ,Biosolids ,Paramagnetic NMR ,Resolution ,Sensitivity ,Structural Biology ,03 medical and health sciences ,Paramagnetism ,Nickel ,Metalloproteins ,Organometallic Compounds ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,Electronic properties ,chemistry.chemical_classification ,0303 health sciences ,Biomolecule ,030302 biochemistry & molecular biology ,Characterization (materials science) ,Solid-state nuclear magnetic resonance ,chemistry ,Metals ,visual_art ,visual_art.visual_art_medium ,Copper - Abstract
Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.
- Published
- 2019
25. Reviewing the Crystal Structure of S100Z and Other Members of the S100 Family: Implications in Calcium-Regulated Quaternary Structure
- Author
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Vito Calderone, Marco Fragai, and Claudio Luchinat
- Subjects
0301 basic medicine ,EF hand ,Supramolecular chemistry ,chemistry.chemical_element ,Computational biology ,Crystal structure ,Calcium ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Sequence homology ,Order (biology) ,chemistry ,030220 oncology & carcinogenesis ,Protein quaternary structure ,Molecular replacement - Abstract
This paper takes the cue from the previously solved crystal structure of human apo-S100Z and compares it with that of the calcium-bound S100Z from zebrafish in order to stress, for this particular S100, the significant role of the presence of calcium in promoting supramolecular assemblies with likely biological meaning. This consideration is then expanded through a wider review on analogous situations concerning all other S100s for which there is crystallographic o biochemical evidence of how the presence of calcium promotes the formation of quaternary complexes.The paper also deals with some considerations on the quality of the crystals obtained for the solved members of this family and on the need for experimental phasing for solving some of the structures where the good general sequence homology among the members of the family would have suggested molecular replacement (MR) as the easiest way to solve them.These considerations, along with the PCA analysis carried out on all the known S100s, further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation for several members of this family of proteins.
- Published
- 2019
26. Interaction of Half Oxa-/Half cis-Platin Complex with Human Superoxide Dismutase and Induced Reduction of Neurotoxicity
- Author
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Lucia Banci, Vito Calderone, Cristina Nativi, L. Di Cesare Mannelli, Francesca Cantini, Leonardo Gonnelli, Carla Ghelardini, M. Korsak, and Oscar Francesconi
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,biology ,Chemistry ,Stereochemistry ,Organic Chemistry ,Neurotoxicity ,Protein aggregation ,medicine.disease ,Biochemistry ,In vitro ,Superoxide dismutase ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,In vivo ,Oxidoreductase ,Drug Discovery ,Toxicity ,biology.protein ,medicine ,030217 neurology & neurosurgery ,Cysteine - Abstract
[Image: see text] The formation of amorphous protein aggregates containing human superoxide dismutase (hSOD1) is thought to be involved in amyotrophic lateral sclerosis onset. cis-Platin inhibits the oligomerization of apo hSOD1, but its toxicity precludes any possible use in therapy. Herein, we propose a less toxic platinum complex, namely oxa/cis-platin, as hSOD1 antiaggregation lead compound. Oxa/cis-platin is able to interact with hSOD1 in the disulfide oxidized apo form by binding cysteine 111 (Cys111). The mild neurotoxic phenomena induced in vitro and in vivo by oxa/cis-platin can be successfully reverted by using lypoyl derivatives, which do not interfere with the antiaggregation properties of the platin derivative.
- Published
- 2018
27. In-house high-energy-remote SAD phasing using the magic triangle: how to tackle the P1 low symmetry using multiple orientations of the same crystal of human IBA57 to increase the multiplicity
- Author
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Lucia Banci, Vito Calderone, Simone Ciofi-Baffoni, Spyridon Gourdoupis, and Veronica Nasta
- Subjects
0301 basic medicine ,Models, Molecular ,Computer science ,Protein Conformation ,Triclinic crystal system ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,03 medical and health sciences ,Software ,Structural Biology ,Humans ,Multiplicity (chemistry) ,Data processing ,business.industry ,SAD phasing ,I3C ,magic triangle ,P1 ,IBA57 ,Phaser ,0104 chemical sciences ,030104 developmental biology ,Substructure ,Raw data ,business ,Carrier Proteins ,Crystallization ,Model building ,Algorithm - Abstract
This article describes the approach used to solve the structure of human IBA57 in-house by 5-amino-2,4,6-triiodoisophthalic acid (I3C) high-energy-remote single-wavelength anomalous dispersion (SAD) phasing. Multiple orientations of the same triclinic crystal were exploited to acquire sufficient real data multiplicity for phasing. How the collection of an in-house native data set and its joint use with the I3C derivative through a SIRAS approach decreases the data multiplicity needed by almost 50% is described. Furthermore, it is illustrated that there is a clear data-multiplicity threshold value for success and failure in phasing, and how adding further data does not significantly affect substructure solution and model building. To our knowledge, this is the only structure present in the PDB that has been solved in-house by remote SAD phasing in space group P1 using only one crystal. All of the raw data used, derived from the different orientations, have been uploaded to Zenodo in order to enable software developers to improve methods for data processing and structure solution, and for educational purposes.
- Published
- 2018
28. IBA57 Recruits ISCA2 to Form a [2Fe-2S] Cluster-Mediated Complex
- Author
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Spyridon Gourdoupis, Vito Calderone, Veronica Nasta, Lucia Banci, and Simone Ciofi-Baffoni
- Subjects
0301 basic medicine ,030102 biochemistry & molecular biology ,Protein family ,Chemistry ,IBA57, iron-sulphur cluster ,General Chemistry ,Oxidative phosphorylation ,Plasma protein binding ,Biochemistry ,Aconitase ,Catalysis ,In vitro ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Colloid and Surface Chemistry ,GLRX5 ,Cluster (physics) ,Cysteine - Abstract
The maturation of mitochondrial iron-sulfur proteins requires a complex protein machinery. Human IBA57 protein was proposed to act in a late phase of this machinery, along with GLRX5, ISCA1, and ISCA2. However, a molecular picture on how these proteins cooperate is not defined yet. We show here that IBA57 forms a heterodimeric complex with ISCA2 by bridging a [2Fe-2S] cluster, that [2Fe-2S] cluster binding is absolutely required to promote the complex formation, and that the cysteine of the conserved motif characterizing IBA57 protein family and the three conserved cysteines of the ISCA protein family act as cluster ligands. The [2Fe-2S] heterodimeric complex is the final product when IBA57 is either exposed to [2Fe-2S] ISCA2 or in the presence of [2Fe-2S] GLRX5 and apo ISCA2. We also find that the [2Fe-2S] ISCA2-IBA57 complex is resistant to highly oxidative environments and is capable of reactivating apo aconitase in vitro. Collectively, our data delinate a [2Fe-2S] cluster transfer pathway involving three partner proteins of the mitochondrial ISC machinery, that is, GLRX5, ISCA2 and IBA57, which leads to the formation of a [2Fe-2S] ISCA2-IBA57 complex.
- Published
- 2018
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29. Human Ind1 expression causes over-expression of E. coli beta-lactamase ampicillin resistance protein
- Author
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Maciej Mikolajczyk, Vaishali Sharma Chakrabarti, Francesca Boscaro, and Vito Calderone
- Subjects
Iron-Sulfur Proteins ,Models, Molecular ,Untranslated region ,Rossmann fold ,Protein Conformation ,Iron–sulfur cluster ,Biology ,Mitochondrion ,beta-Lactamases ,Mitochondria ,Mitochondrial Proteins ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Cyclic nucleotide-binding domain ,Escherichia coli ,Humans ,Homology modeling ,Inner mitochondrial membrane ,Ampicillin Resistance ,Biogenesis ,Biotechnology - Abstract
Ind1, a mitochondrial P-loop NTPase is essential for assembly of respiratory complex-I. Respiratory complex-I (NADH: ubiquinone oxidoreductase), a large (mitochondrial inner membrane) enzyme, is made of 45 subunits and 8 iron-sulfur clusters. Ind1, an iron-sulfur cluster protein involved in the maturation of respiratory complex and binds an Fe/S cluster via a conserved CXXC motif in a labile way. Ind1 has been proposed as a specialized biogenesis factor involved in delivering the Fe/S clusters to the apo complex-I subunits. The IND1 gene is conserved in eukaryotes and is present in genomes of the species that retain functional respiratory complex-I. Depletion of human Ind1 causes ultra-structural changes in depleted mitochondria, including the loss of cristae membranes, massive remodeling of respiratory super complexes, and increased lactate production. Ind1 sequence bears known nucleotide binding domain motifs and was first classified as Nucleotide Binding Protein-Like (NUBPL). Despite the obvious importance of Ind1, very little is known about this protein; in particular its structure as well as its Fe/S cluster binding properties. In the present work we show that the expression of native huInd1 in Escherichia coli stimulates over-expression of the beta-lactamase TEM-1 from E. coli. The homology modeling of huInd1 shows hallmark of Rossmann fold, where a central beta sheet is covered by helices on either side. In the light of the modeled structure of huInd1, we hypothesize that huInd1 binds to the untranslated region (UTR) of the TEM-1 mRNA at 3' site and thereby reducing the possibility of its endonucleolytic cleavage, resulting in over-expression of TEM-1.
- Published
- 2014
30. Simultaneous use of solution NMR and X-ray data inREFMAC5 for joint refinement/detection of structural differences
- Author
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Mauro Rinaldelli, Vito Calderone, Claudio Luchinat, Giacomo Parigi, Enrico Ravera, and Garib N. Murshudov
- Subjects
Models, Molecular ,Calmodulin ,structure refinement ,Crystallography, X-Ray ,RDC ,010402 general chemistry ,Residual ,01 natural sciences ,X-ray ,03 medical and health sciences ,Paramagnetism ,REFMAC ,Structural Biology ,Catalytic Domain ,Atomic model ,Molecule ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,Coupling ,0303 health sciences ,biology ,Chemistry ,Proteins ,Reproducibility of Results ,General Medicine ,Research Papers ,0104 chemical sciences ,Crystallography ,Experimental uncertainty analysis ,PCS ,biology.protein ,Diamagnetism - Abstract
Paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and diamagnetic residual dipolar couplings can now be used in the program REFMAC5 from CCP4 as structural restraints together with X-ray crystallographic data. These NMR restraints can reveal differences between solid state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement., The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably ‘ideal’ geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.
- Published
- 2014
31. Molecular Determinants of a Selective Matrix Metalloprotease-12 Inhibitor: Insights from Crystallography and Thermodynamic Studies
- Author
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Claudio Luchinat, Bertrand Czarny, Evelyne Cassar-Lajeunesse, Laurent Devel, Vito Calderone, Enrico A. Stura, Fabrice Beau, Laura Vera, Vincent Dive, and Marco Fragai
- Subjects
Models, Molecular ,biology ,Hydrogen bond ,Chemistry ,Stereochemistry ,Active site ,Isothermal titration calorimetry ,Crystal structure ,Matrix metalloproteinase ,Crystallography, X-Ray ,Crystallography ,Matrix Metalloproteinase 12 ,Drug Discovery ,Mole ,Side chain ,biology.protein ,Thermodynamics ,Molecular Medicine ,Protease Inhibitors ,Chelation - Abstract
The molecular determinants responsible for the potency of the RXP470.1 phosphinic peptide inhibitor toward matrix metalloprotease-12 (MMP-12) remain elusive. To address this issue, structure-activity study, X-ray crystallography, and isothermal titration calorimetry (ITC) experiments were performed. The crystal structure of MMP-12/inhibitor complex (1.15 Å) reveals that the inhibitor establishes multiple interactions with the MMP-12 active site, with its long P(1)' side chain filling most of the S(1)' deep cavity. ITC experiments indicate that the binding of this inhibitor to MMP-12 is mostly entropy driven (ΔG° = -13.1 kcal/mol, ΔH° = -2.53 kcal/mol, and -TΔS° = -10.60 kcal/mol) and involves a proton uptake from the buffer. Comparing phosphinic versus hydroxamate inhibitors reveals that the chelation of the zinc ion is slightly different, leading the inhibitor backbone to adopt a position in which the hydrogen bonding with the MMP-12 active site is less favorable in phosphinic inhibitor while maintaining high affinity.
- Published
- 2013
32. Structure of the extended-spectrum β-lactamase TEM-72 inhibited by citrate
- Author
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Gian Maria Rossolini, Jean Denis Docquier, Vito Calderone, Stefano Mangani, and Manuela Benvenuti
- Subjects
Models, Molecular ,crystal structure ,Stereochemistry ,medicine.medical_treatment ,Molecular Sequence Data ,Mutant ,Biophysics ,Biochemistry ,Citric Acid ,beta-Lactamases ,Serine ,chemistry.chemical_compound ,Structural Biology ,Hydrolase ,Genetics ,medicine ,Structural Communications ,x-ray ,beta-lactamase ,tem-72 ,chemistry.chemical_classification ,biology ,Active site ,Condensed Matter Physics ,biology.organism_classification ,Enterobacteriaceae ,Protein Structure, Tertiary ,Enzyme ,chemistry ,biology.protein ,Beta-lactamase ,beta-Lactamase Inhibitors ,Citric acid - Abstract
TEM-72, a class A β-lactamase identified in isolates of Enterobacteriaceae, is a quadruple mutant of TEM-1 (Q39K, M182T, G238S and E240K) and shows extended-spectrum β-lactamase (ESBL) properties arising from the G238S and E240K substitutions. Although many structures of TEM variants have been published, they do not include an enzyme with the simultaneous presence of both of the ESBL-conferring G238S and E240K substitutions. Furthermore, the structure shows the presence of a citrate anion bound to the TEM-72 active site, where it interacts with all of the conserved residues of class A β-lactamases. The present structure supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases.
- Published
- 2011
33. Structure-based approach to nanomolar, water soluble matrix metalloproteinases inhibitors (MMPIs)
- Author
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Barbara Richichi, Claudio Luchinat, Cristina Nativi, Emanuele Attolino, Marco Fragai, Vito Calderone, and Elisa Dragoni
- Subjects
Models, Molecular ,Molecular model ,Matrix metalloproteinase inhibitor ,Matrix Metalloproteinase Inhibitors ,Matrix metalloproteinase ,Crystallography, X-Ray ,Structure-Activity Relationship ,chemistry.chemical_compound ,Residue (chemistry) ,Drug Discovery ,Structure–activity relationship ,Protease Inhibitors ,Sulfones ,Pharmacology ,chemistry.chemical_classification ,Hydroxamic acid ,Molecular Structure ,biology ,Organic Chemistry ,Water ,Stereoisomerism ,General Medicine ,Nanostructures ,Enzyme ,Solubility ,chemistry ,Biochemistry ,Enzyme inhibitor ,biology.protein - Abstract
N-arylsulfonyl-based MMPs inhibitors (MMPIs) are among the most prominent inhibitors possessing nanomolar affinity. However, their poor bioavailability remains critical for the drug development of this family of molecules. The structural analysis of the complex of NNGH (the most representative member of the family) with MMP-12 provided us with the basis to effectively design simple NNGH analogues with enhanced solubility in water. Following this approach, the sec-butyl residue, not directly involved in the binding with MMP, has been replaced with hydrophilic residues thus yielding new potent inhibitors soluble in water.
- Published
- 2010
34. Copper(I)-mediated protein–protein interactions result from suboptimal interaction surfaces
- Author
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Sara Neri, Antonio Rosato, Vito Calderone, Anna Pavelkova, Lucia Banci, Isabella C. Felli, Ivano Bertini, Nunzia Della-Malva, Magnetic Resonance Center, and Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI)
- Subjects
Surface Properties ,ATPase ,Static Electricity ,ATP7A ,chemistry.chemical_element ,Plasma protein binding ,Protein Serine-Threonine Kinases ,Crystallography, X-Ray ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Adduct ,Protein–protein interaction ,ATOX1 ,03 medical and health sciences ,Copper Transport Proteins ,Humans ,Cation Transport Proteins ,Molecular Biology ,030304 developmental biology ,Adenosine Triphosphatases ,0303 health sciences ,biology ,Intracellular Signaling Peptides and Proteins ,Life Sciences ,Cell Biology ,Copper ,Protein Structure, Tertiary ,0104 chemical sciences ,Metallochaperones ,Solutions ,Crystallography ,chemistry ,Copper-Transporting ATPases ,Mutation ,Copper-transporting ATPases ,biology.protein ,Biophysics ,Cadmium ,Molecular Chaperones ,Protein Binding - Abstract
The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein–protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, α polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 Å2, 1 Å=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.
- Published
- 2009
35. Structural Basis of Serine/Threonine Phosphatase Inhibition by the Archetypal Small Molecules Cantharidin and Norcantharidin
- Author
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Claudio Luchinat, Ivano Bertini, Vito Calderone, E. Talluri, and Marco Fragai
- Subjects
chemistry.chemical_classification ,Cantharidin ,Binding Sites ,Norcantharidin ,Protein Conformation ,Stereochemistry ,Bridged Bicyclo Compounds, Heterocyclic ,Ligand (biochemistry) ,Small molecule ,Serine ,Structure-Activity Relationship ,chemistry.chemical_compound ,Dicarboxylic acid ,chemistry ,Biochemistry ,Drug Discovery ,Phosphoprotein Phosphatases ,Molecular Medicine ,Enzyme Inhibitors ,Threonine ,Binding site ,Hydrophobic and Hydrophilic Interactions - Abstract
The inhibition of a subgroup of human serine/threonine protein phosphatases is responsible for the cytotoxicity of cantharidin and norcantharidin against tumor cells. It is shown that the anhydride rings of cantharidin and norcantharidin are hydrolyzed when bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules are reported. Norcantharidin shows a unique binding conformation with the catalytically active Mn2PP5c, while cantharidin is characterized by a double conformation in its binding mode to the protein. Different binding modes of norcantharidin are observed depending of whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures will provide the basis for the rational design of new cantharidin-based drugs.
- Published
- 2009
36. Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants
- Author
-
Miguela Vieru, Mirela Boca, Ivano Bertini, Stefania Girotto, Lucia Banci, Francesca Cantini, and Vito Calderone
- Subjects
Models, Molecular ,Stereochemistry ,SOD1 ,Crystal structure ,Crystallography, X-Ray ,Protein Structure, Secondary ,law.invention ,Crystal ,Superoxide Dismutase-1 ,Protein structure ,law ,Oxidoreductase ,Humans ,Crystallization ,Protein Structure, Quaternary ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Multidisciplinary ,Superoxide Dismutase ,Chemistry ,Biological Sciences ,Protein Structure, Tertiary ,Solvent ,Crystallography ,Mutation ,Protein Multimerization ,Apoproteins ,Cysteine - Abstract
The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.
- Published
- 2009
37. Evidence of Reciprocal Reorientation of the Catalytic and Hemopexin-Like Domains of Full-Length MMP-12
- Author
-
Dmitri I. Svergun, Efstratios Mylonas, Marco Fragai, Vito Calderone, Maxime Melikian, Claudio Luchinat, Rahul Jaiswal, and Ivano Bertini
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Chemokine ,biology ,Protein Conformation ,Chemistry ,Stereochemistry ,Hemopexin ,General Chemistry ,Matrix metalloproteinase ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Extracellular matrix ,Colloid and Surface Chemistry ,Enzyme ,Cell surface receptor ,Catalytic Domain ,Matrix Metalloproteinase 12 ,Hydrolase ,biology.protein ,Biophysics ,Nuclear Magnetic Resonance, Biomolecular ,Linker - Abstract
The proteolytic activity of matrix metalloproteinases toward extracellular matrix components (ECM), cytokines, chemokines, and membrane receptors is crucial for several homeostatic and pathological processes. Active MMPs are a family of single-chain enzymes (23 family members in the human genome), most of which constituted by a catalytic domain and by a hemopexin-like domain connected by a linker. The X-ray structures of MMP-1 and MMP-2 suggest a conserved and well-defined spatial relationship between the two domains. Here we present structural data for MMP-12, suitably stabilized against self-hydrolysis, both in solution (NMR and SAXS) and in the solid state (X-ray), showing that the hemopexin-like and the catalytic domains experience conformational freedom with respect to each other on a time scale shorter than 10(-8) s. Hints on the probable conformations are also obtained. This experimental finding opens new perspectives for the often hypothesized active role of the hemopexin-like domain in the enzymatic activity of MMPs.
- Published
- 2008
38. Exploring the Subtleties of Drug−Receptor Interactions: The Case of Matrix Metalloproteinases
- Author
-
Cristina Nativi, Ivano Bertini, Mauro Loconte, Kwon Joo Yeo, Andrea Giachetti, Claudio Luchinat, Marco Fragai, M. Maletta, and Vito Calderone
- Subjects
Stereochemistry ,Receptors, Drug ,Matrix Metalloproteinase Inhibitors ,Hydroxamic Acids ,Ligands ,Biochemistry ,Catalysis ,Adduct ,Colloid and Surface Chemistry ,Matrix Metalloproteinase 12 ,Matrix Metalloproteinase 13 ,Hydrolase ,medicine ,Drug Interactions ,Protease Inhibitors ,Binding Sites ,Chemistry ,Drug discovery ,Acetohydroxamic acid ,Titrimetry ,Isothermal titration calorimetry ,General Chemistry ,Ligand (biochemistry) ,Thermodynamics ,Drug receptor ,Selectivity ,medicine.drug - Abstract
By solving high-resolution crystal structures of a large number (14 in this case) of adducts of matrix metalloproteinase 12 (MMP12) with strong, nanomolar, inhibitors all derived from a single ligand scaffold, it is shown that the energetics of the ligand-protein interactions can be accounted for directly from the structures to a level of detail that allows us to rationalize for the differential binding affinity between pairs of closely related ligands. In each case, variations in binding affinities can be traced back to slight improvements or worsening of specific interactions with the protein of one or more ligand atoms. Isothermal calorimetry measurements show that the binding of this class of MMP inhibitors is largely enthalpy driven, but a favorable entropic contribution is always present. The binding enthalpy of acetohydroxamic acid (AHA), the prototype zinc-binding group in MMP drug discovery, has been also accurately measured. In principle, this research permits the planning of either improved inhibitors, or inhibitors with improved selectivity for one or another MMP. The present analysis is applicable to any drug target for which structural information on adducts with a series of homologous ligands can be obtained, while structural information obtained from in silico docking is probably not accurate enough for this type of study.
- Published
- 2007
39. A hint for the function of human Sco1 from different structures
- Author
-
Shenlin Wang, Lucia Banci, Peep Palumaa, Stefano Mangani, Manuele Martinelli, Ivano Bertini, Vito Calderone, and Simone Ciofi-Baffoni
- Subjects
Models, Molecular ,crystal structure ,Coordination sphere ,XAS ,Electrospray ionization ,Molecular Sequence Data ,Crystallography, X-Ray ,Thioredoxin fold ,Metal ,Residue (chemistry) ,x-ray ,NMR ,Sco1 ,copper chaperone ,Nickel ,Humans ,Amino Acid Sequence ,Multidisciplinary ,biology ,Chemistry ,Ligand ,Membrane Proteins ,Crystallography ,visual_art ,Chaperone (protein) ,Physical Sciences ,visual_art.visual_art_medium ,biology.protein ,Thioredoxin ,Apoproteins ,Copper ,Molecular Chaperones - Abstract
The solution structures of apo, Cu(I), and Ni(II) human Sco1 have been determined. The protein passes from an open and conformationally mobile state to a closed and rigid conformation upon metal binding as shown by electrospray ionization MS and NMR data. The metal ligands of Cu(I) are two Cys residues of the CPXXCP motif and a His residue. The latter is suitably located to coordinate the metal anchored by the two Cys residues. The coordination sphere of Ni(II) in solution is completed by another ligand, possibly Asp. Crystals of the Ni(II) derivative were also obtained with the Ni(II) ion bound to the same His residue and to the two oxidized Cys residues of the CPXXCP motif. We propose that the various structures solved here represent the various states of the protein in its functional cycle and that the metal can be bound to the oxidized protein at a certain stage. Although it now seems reasonable that Sco1, which is characterized by a thioredoxin fold, has evolved to bind a metal atom via the di-Cys motif to act as a copper chaperone, the oxidized form of the nickel-bound protein suggests that it may also maintain the thioredoxin function.
- Published
- 2006
40. The crystal structure of yeast copper thionein: The solution of a long-lasting enigma
- Author
-
Vito Calderone, Benedikt Dolderer, Hartmut Echner, Stefano Mangani, Claudio Luchinat, Ulrich Weser, Cristina Del Bianco, and Hans-Juergen Dr Hartmann
- Subjects
Metal ions in aqueous solution ,chemistry.chemical_element ,Saccharomyces cerevisiae ,Crystal structure ,Crystallography, X-Ray ,Metal ,Protein structure ,Yeasts ,Metallothionein ,Multidisciplinary ,Chemistry ,Biological Sciences ,metallothionein ,x-ray structure ,Copper ,copper metabolism ,Random coil ,Protein Structure, Tertiary ,Crystallography ,visual_art ,visual_art.visual_art_medium ,Crystallization ,Protein Binding ,Cysteine - Abstract
We report here the crystal structure of yeast copper thionein (Cu-MT), determined at 1.44-Å resolution. The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biology, consisting of six trigonally and two digonally coordinated Cu(I) ions. This is at variance with the results from previous spectroscopic determinations, which were performed on MT samples containing seven rather than eight metal ions. The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues. The protein structure is somewhat different from that of Ag 7 -MT and similar, but not identical, to that of Cu 7 -MT. Besides the different structure of the metal cluster, the main differences lie in the cysteine topology and in the conformation of some portions of the backbone. The present structure suggests that Cu-MT, in addition to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.
- Published
- 2004
41. Crystal Structure of the Catalytic Domain of Human Matrix Metalloproteinase 10
- Author
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Vito Calderone, Marco Fragai, Stefano Mangani, Beatrice Terni, Claudio Luchinat, and Ivano Bertini
- Subjects
Models, Molecular ,crystal structure ,matrix metalloproteinase ,Stereochemistry ,Molecular Sequence Data ,Crystal structure ,Matrix (biology) ,Matrix metalloproteinase ,Crystallography, X-Ray ,medicine.disease_cause ,Catalysis ,chemistry.chemical_compound ,Matrix Metalloproteinase 10 ,Structural Biology ,stromelysin-2 ,Catalytic Domain ,Hydrolase ,medicine ,Humans ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,Hydroxamic acid ,Molecular Structure ,Chemistry ,Metalloendopeptidases ,matrix metalloproteinase, stromelysin-2, MMP-10, crystal structure, inhibitor ,Protein Structure, Tertiary ,inhibitor ,Stromelysin-2 ,MMP-10 ,Sequence Alignment - Abstract
The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.
- Published
- 2004
42. Crystal structure of the PsbQ protein of photosystem II from higher plants
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Flora Andreucci, Roberto Battistutta, Michela Trabucco, Andreja Vujičić, Giorgio M. Giacometti, Vito Calderone, Giuseppe Zanotti, and Roberto Barbato
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Spinacia ,Photosystem II ,Scientific Report ,Amino Acid Motifs ,Molecular Sequence Data ,chemistry.chemical_element ,macromolecular substances ,Zinc ,Crystal structure ,Crystallography, X-Ray ,Photosystem I ,Biochemistry ,Crystal ,Spinacia oleracea ,Genetics ,Amino Acid Sequence ,Molecular Biology ,Photosystem ,Binding Sites ,biology ,Arabidopsis Proteins ,Photosystem II Protein Complex ,food and beverages ,biology.organism_classification ,Protein Structure, Tertiary ,Crystallography ,chemistry ,Spinach - Abstract
The smallest extrinsic polypeptide of the water-oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn2+ and its structure was determined by X-ray diffraction at 1.95-Å resolution using the multi-wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four-helix bundle, whereas the amino-terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl− and Ca2+ and make them available to PSII.
- Published
- 2003
43. Identification and Structural Analysis of a Zebrafish Apo and Holo Cellular Retinol-binding Protein
- Author
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Claudia Folli, Vito Calderone, Alessio Marchesani, Giuseppe Zanotti, and Rodolfo Berni
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Protein Conformation ,Molecular Sequence Data ,macromolecular substances ,Biology ,Crystallography, X-Ray ,environment and public health ,DNA-binding protein ,chemistry.chemical_compound ,Structural Biology ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Zebrafish ,DNA Primers ,Base Sequence ,Sequence Homology, Amino Acid ,Retinol ,Retinol-Binding Proteins, Cellular ,Ligand (biochemistry) ,biology.organism_classification ,Rats ,Transport protein ,Retinol-Binding Proteins ,Biochemistry ,chemistry ,Cytoplasm ,Retinol binding - Abstract
Cellular retinol-binding proteins (CRBPs) are cytoplasmic retinol-specific binding proteins. Mammalian CRBPs have been thoroughly characterised previously. Here we report on the identification and X-ray structural analysis of the apo (1.7A resolution) and holo (1.4A resolution) forms of a zebrafish CRBP. According to amino acid sequence and structure analyses, the zebrafish CRBP that we have identified resembles closely mammalian CRBP II, suggesting that it is the zebrafish orthologue of this mammalian CRBP type. Zebrafish CRBP forms a tight complex with all-trans retinol, producing an absorption spectrum similar to those of mammalian holo-CRBPs, albeit slightly blue-shifted. The superposition of the alpha-carbon atoms of the liganded (complexed with retinol) and unliganded forms of zebrafish CRBP shows significant differences in correspondence of the betaC-betaD (residues 55-58) and betaE-betaF (residues 74-77) turns, providing evidence for the occurrence of conformational changes accompanying retinol binding/release. Remarkable and well-defined ligand-dependent conformational changes in the protein region comprising the two beta-turns affect both the main chain and the side-chains of several residues. The two beta-turns project towards the interior of the cavity devoid of ligand of the apoprotein. The side-chains of F57, Y60 and L77 change substantially their orientation and position in the apoprotein relative to the holoprotein. In the beta-barrel internal cavity of apo-CRBP they occupy some of the space that is otherwise occupied by bound retinol in holo-CRBP, and are displaced from these positions on ligand binding. These results indicate that a flexible area encompassing the betaC-betaD and betaE-betaF turns may serve as the ligand portal and that these turns undergo conformational changes associated with the not yet clarified mechanism of retinol binding and release in CRBPs.
- Published
- 2002
44. The Unusual Amino Acid Triplet Asn–Ile–Cys Is a Glycosylation Consensus Site in Human α-Lactalbumin
- Author
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Annamaria Cantisani, Jasminka Godovac-Zimmermann, Amedeo Conti, Vito Calderone, Maria Gabriella Giuffrida, Ben Neuteboom, Carlo Giunta, Maria Cavaletto, and Lorenzo Napolitano
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Glycosylation ,animal structures ,Consensus site ,Molecular Sequence Data ,macromolecular substances ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,Protein sequencing ,Humans ,Bioorganic chemistry ,Amino Acid Sequence ,Conserved Sequence ,chemistry.chemical_classification ,Binding Sites ,biology ,Chemistry ,Molten globule ,Amino acid ,carbohydrates (lipids) ,Lactalbumin ,Alpha-lactalbumin ,biology.protein ,Female ,lipids (amino acids, peptides, and proteins) ,Glycoprotein ,Oligopeptides ,Sequence Analysis - Abstract
Human alpha-lactalbumin has not been described as a glycoprotein, despite the fact that several alpha-lactalbumins of both ruminant and nonruminant species are known to be glycosylated. In all these species the glycosylation site is the 45Asn in the usual triplet 45Asn-Gly/Gln-47Ser. We have found that human alpha-lactalbumin is glycosylated and the glycosylation site has been determined by protein sequencing and mass spectrometry. We report an unusual glycosylation site at 71Asn in the triplet 71Asn-Ile-73Cys, which is conserved in all known alpha-lactalbumins except red-necked wallaby. That a relatively small proportion of the protein is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of alpha-lactalbumin.
- Published
- 1997
45. Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis
- Author
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Mario Piccioli, Andrea Giachetti, Ivano Bertini, Maciej Mikolajczyk, Julia Winkelmann, Lucia Banci, Vito Calderone, Simone Ciofi-Baffoni, and Deepa Jaiswal
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Iron-Sulfur Proteins ,Models, Molecular ,Molecular model ,Flavin Mononucleotide ,Plasma protein binding ,Biology ,010402 general chemistry ,01 natural sciences ,Redox ,Models, Biological ,Electron Transport ,03 medical and health sciences ,Electron transfer ,Oxidoreductase ,Protein Interaction Mapping ,Protein biosynthesis ,Humans ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Flavoproteins ,Intracellular Signaling Peptides and Proteins ,Electron transport chain ,0104 chemical sciences ,Cell biology ,Protein Structure, Tertiary ,chemistry ,Protein Biosynthesis ,Physical Sciences ,Oxidoreductases ,Biogenesis ,Protein Binding - Abstract
Biogenesis of iron–sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron–sulfur protein assembly machinery, two human key proteins—NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin—form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron–sulfur cluster proteins. The Ndor1–anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.
- Published
- 2013
46. Discovery of a New Class of Potent MMP Inhibitors by Structure-Based Optimization of the Arylsulfonamide Scaffold
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Assunta Massaro, Alessandro Mordini, Vito Calderone, Claudio Luchinat, Mattia Mori, and Marco Fragai
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chemistry.chemical_classification ,mmp ,Chemistry ,metalloproteins ,molecular association ,Organic Chemistry ,drug ,structure activity relationship ,Matrix metalloproteinase ,Biochemistry ,Combinatorial chemistry ,chemistry.chemical_compound ,Residue (chemistry) ,Docking (molecular) ,Drug Discovery ,Metalloprotein ,Moiety ,Carboxylate ,Selectivity ,Linker - Abstract
A new class of potent matrix metalloproteinase (MMP) inhibitors designed by structure-based optimization of the well-known arylsulfonamide scaffold is presented. Molecules show an ethylene linker connecting the sulfonamide group with the P1′ aromatic portion and a d-proline residue bearing the zinc-binding group. The affinity improvement provided by these modifications led us to discover a nanomolar MMP inhibitor bearing a carboxylate moiety as zinc-binding group, which might be a promising lead molecule. Notably, a significant selectivity for MMP-8, MMP-12, and MMP-13 was observed with respect to MMP-1 and MMP-7.
- Published
- 2012
47. Interaction of cisplatin with human superoxide dismutase
- Author
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Olga Blaževitš, Miguela Vieru, Ilaria Amori, Jiafei Mao, Vito Calderone, Ivano Bertini, Francesca Cantini, Angela Trapananti, Lucia Banci, Maria Teresa Carrì, and Mauro Cozzolino
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Models, Molecular ,inorganic chemicals ,Stereochemistry ,Protein subunit ,cisplatin ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Cell Line ,Superoxide dismutase ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,Colloid and Surface Chemistry ,Oxidoreductase ,medicine ,Animals ,Humans ,Structure–activity relationship ,Enzyme Inhibitors ,Settore BIO/10 ,030304 developmental biology ,Cisplatin ,chemistry.chemical_classification ,human superoxide dismutase ,0303 health sciences ,biology ,Chemistry ,General Chemistry ,superoxide dismutase ,In vitro ,0104 chemical sciences ,3. Good health ,Solutions ,Dissociation constant ,superoxide dismutase, cisplatin ,biology.protein ,medicine.drug ,Cysteine - Abstract
cis-Diamminedichloroplatinum(II) (cisplatin) is able to interact with human superoxide dismutase (hSOD1) in the disulfide oxidized apo form with a dissociation constant of 37 ± 3 μM through binding cysteine 111 (Cys111) located at the edge of the subunit interface. It also binds to Cu(2)-Zn(2) and Zn(2)-Zn(2) forms of hSOD1. Cisplatin inhibits aggregation of demetalated oxidized hSOD1, and it is further able to dissolve and monomerize oxidized hSOD1 oligomers in vitro and in cell, thus indicating its potential as a leading compound for amyotrophic lateral sclerosis.
- Published
- 2012
48. The catalytic domain of MMP-1 studied through tagged lanthanides
- Author
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Carlos F. G. C. Geraldes, Ivano Bertini, Linda Cerofolini, João M.C. Teixeira, Petr Hermann, Claudio Luchinat, Vito Calderone, Marco Fragai, and Giacomo Parigi
- Subjects
Lanthanide ,Models, Molecular ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Biophysics ,Residual dipolar coupling ,Crystal structure ,010402 general chemistry ,Crystallography, X-Ray ,01 natural sciences ,Biochemistry ,Lanthanoid Series Elements ,Protein Structure, Secondary ,Paramagnetism ,Protein structure ,Structural Biology ,Catalytic Domain ,Hydrolase ,Paramagnetic tag ,Genetics ,Molecular Biology ,Protein secondary structure ,Paramagnetic restraint ,010405 organic chemistry ,Chemistry ,Cell Biology ,0104 chemical sciences ,Solutions ,Matrix metalloproteinase ,Crystallography ,Domain (ring theory) ,Matrix Metalloproteinase 1 - Abstract
Pseudocontact shifts (pcs) and paramagnetic residual dipolar couplings (rdc) provide structural information that can be used to assess the adequacy of a crystallographic structure to represent the solution structure of a protein. This can be done by attaching a lanthanide binding tag to the protein. There are cases in which only local rearrangements are sufficient to match the NMR data and cases where significant secondary structure or domain rearrangements from the solid state to the solution state are needed. We show that the two cases are easily distinguishable. Whereas the use of solution restraints in the latter case is described in the literature, here we deal with how to obtain a better model of the solution structure in a case (the catalytic domain of the matrix metalloproteinase MMP-1) of the former class.
- Published
- 2012
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49. An electron-transfer path through an extended disulfide relay system: the case of the redox protein ALR
- Author
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Ivano Bertini, Lucia Banci, Kostas Tokatlidis, Simone Ciofi-Baffoni, Vito Calderone, Angelo Gallo, and Chiara Cefaro
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Models, Molecular ,Cytochrome ,LIVER-REGENERATION ,Flavoprotein ,Photochemistry ,Mitochondrial Membrane Transport Proteins ,Biochemistry ,Redox ,Catalysis ,Electron Transport ,MITOCHONDRIAL INTERMEMBRANE SPACE ,03 medical and health sciences ,Electron transfer ,EGG-WHITE ,0302 clinical medicine ,Colloid and Surface Chemistry ,SUBSTRATE ,Thiol oxidase ,Mitochondrial Precursor Protein Import Complex Proteins ,Oxidoreductases Acting on Sulfur Group Donors ,Disulfides ,Cytochrome Reductases ,030304 developmental biology ,0303 health sciences ,biology ,AUGMENTOR ,Chemistry ,Circular Dichroism ,Cytochrome c ,Oxidative folding ,IMPORT ,Cytochromes c ,General Chemistry ,DEPENDENT SULFHYDRYL OXIDASES ,BOND FORMATION ,FLAVIN ,MIA40 ,biology.protein ,Biophysics ,Spectrophotometry, Ultraviolet ,Intermembrane space ,Oxidation-Reduction ,030217 neurology & neurosurgery - Abstract
The oxidative folding mechanism in the intermembrane space of human mitochondria underpins a disulfide relay system consisting of the import receptor Mia40 and the homodimeric FAD-dependent thiol oxidase ALR. The flavoprotein ALR receives two electrons per subunit from Mia40, which are then donated through one-electron reactions to two cytochrome c molecules, thus mediating a switch from two-electron to one-electron transfer. We dissect here the mechanism of the electron flux within ALR, characterizing at the atomic level the ALR intermediates that allow electrons to rapidly flow to cytochrome c. The intermediate critical for the electron-transfer process implies the formation of a specific inter-subunit disulfide which exclusively allows electron flow from Mia40 to FAD. This finding allows us to present a complete model for the electron-transfer pathway in ALR.
- Published
- 2012
50. Structural characterization of human S100A16, a low-affinity calcium binder
- Author
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Giacomo Parigi, Elena Babini, Claudio Luchinat, Ivano Bertini, Valentina Borsi, Xiaoyu Hu, Vito Calderone, E. Babini, I. Bertini, V. Borsi, V. Calderone, X. Hu, C. Luchinat, and G. Parigi
- Subjects
Magnetic Resonance Spectroscopy ,Molecular Sequence Data ,chemistry.chemical_element ,EFHAND ,Calorimetry ,Calcium ,Biochemistry ,Protein Structure, Secondary ,Inorganic Chemistry ,Hydrophobic effect ,Calcium-binding protein ,Side chain ,Humans ,Amino Acid Sequence ,Binding site ,Chemistry ,Protein dynamics ,Calcium-Binding Proteins ,S100 Proteins ,Cooperative binding ,NMR ,S100A16 ,Crystallography ,CALCIUM BINDING PROTEINS ,Binding domain ,S100 - Abstract
The homodimeric structure of human S100A16 in the apo state has been obtained both in the solid state and in solution, resulting in good agreement between the structures with the exception of two loop regions. The homodimeric solution structure of human S100A16 was also calculated in the calcium(II)-bound form. Differently from most S100 proteins, the conformational rearrangement upon calcium binding is minor. This characteristic is likely to be related to the weak binding affinity of the protein for the calcium(II) ions. In turn, this is ascribed to the lack of the glutamate residue at the end of the S100-specific N-domain binding site, which in most S100 proteins provides two important side chain oxygen atoms as calcium(II) ligands. Furthermore, the presence of hydrophobic interactions stronger than for other S100 proteins, present in the closed form of S100A16 between the third and fourth helices, likely make the closed structure of the second EF-hand particularly stable, so even upon calcium(II) binding such a conformation is not disrupted.
- Published
- 2011
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