Publications
133 results found
Visconti L, Toto A, Jarvis J, et al., 2020, Demonstration of binding induced structural plasticity in a SH2 domain, Frontiers in Molecular Biosciences, Vol: 7, ISSN: 2296-889X
SH2 domains are common protein interaction domains able to recognize short aminoacidic sequences presenting a phosphorylated tyrosine (pY). In spite of their fundamental importance for cell physiology there is a lack of information about the mechanism by which these domains recognize and bind their natural ligands. The N-terminal SH2 (N-SH2) domain of PI3K mediates the interaction with different scaffolding proteins and is known to recognize a specific pY-X-X-M consensus sequence. These interactions are at the cross roads of different molecular pathways and play a key role for cell development and division. By combining mutagenesis, chemical kinetics and NMR, here we provide a complete characterization of the interaction between N-SH2 and a peptide mimicking the scaffolding protein Gab2. Our results highlight that N-SH2 is characterized by a remarkable structural plasticity, with the binding reaction being mediated by a diffused structural region and not solely by the residues located in the binding pocket. Furthermore, the analysis of kinetic data allow us to pinpoint an allosteric network involving residues far from the binding pocket involved in specificity. Results are discussed on the light of previous works on the binding properties of SH2 domains.
Achour A, Broggini L, Han X, et al., 2020, Biochemical and biophysical comparison of human and mouse beta-2 microglobulin reveals the molecular determinants of low amyloid propensity, FEBS JOURNAL, Vol: 287, Pages: 546-560, ISSN: 1742-464X
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- Citations: 8
Man WK, De Simone A, Barritt JD, et al., 2020, A Role of Cholesterol in Modulating the Binding of α-Synuclein to Synaptic-Like Vesicles, FRONTIERS IN NEUROSCIENCE, Vol: 14
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- Citations: 23
Runfola M, De Simone A, Vendruscolo M, et al., 2020, The N-terminal Acetylation of α-Synuclein Changes the Affinity for Lipid Membranes but not the Structural Properties of the Bound State, SCIENTIFIC REPORTS, Vol: 10, ISSN: 2045-2322
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- Citations: 38
Baum J, Chiti F, De Simone A, et al., 2019, Homage to Chris Dobson, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 6
Qu F, ElOmari K, Wagner A, et al., 2019, Desolvation of the substrate binding protein TauA dictates ligand specificity for the alkanesulfonate ABC importer TauABC, Biochemical Journal, Vol: 476, Pages: 3649-3660, ISSN: 0264-6021
Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterised as a taurine specific system. TauA and SsuA are substrate binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterised its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent in SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters.
Wang X, Kirkpatrick JP, Launay HMM, et al., 2019, Probing the dynamic stalk region of the ribosome using solution NMR, SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322
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- Citations: 8
Vivoli Vega M, Cascella R, Chen SW, et al., 2019, The Toxicity of Misfolded Protein Oligomers Is Independent of Their Secondary Structure., ACS Chem Biol, Vol: 14, Pages: 1593-1600
The self-assembly of proteins into structured fibrillar aggregates is associated with a range of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, in which an important cytotoxic role is thought to be played by small soluble oligomers accumulating during the aggregation process or released by mature fibrils. As the structural characteristics of such species and their links with toxicity are still not fully defined, we have compared six examples of preformed misfolded protein oligomers with different β-sheet content, as determined using Fourier transform infrared spectroscopy, and with different toxicity, as determined by three cellular readouts of cell viability. The results show the absence of any measurable correlation between the nature of their secondary structure and their cellular toxicity, both when comparing the six types of oligomers as a group and when comparing species in subgroups characterized by either the same size or the same exposure of hydrophobic moieties.
Cascella R, Perni M, Chen SW, et al., 2019, Probing the Origin of the Toxicity of Oligomeric Aggregates of alpha-Synuclein with Antibodies, ACS CHEMICAL BIOLOGY, Vol: 14, Pages: 1352-1362, ISSN: 1554-8929
He S, Biedermann F, Vankova N, et al., 2018, Cavitation energies can outperform dispersion interactions, Nature Chemistry, Vol: 10, Pages: 1252-1257, ISSN: 1755-4330
The accurate dissection of binding energies into their microscopic components is challenging, especially in solution. Here we study the binding of noble gases (He-Xe) with the macrocyclic receptor cucurbit[5]uril in water by displacement of methane and ethane as 1H NMR probes. We dissect the hydration free energies of the noble gases into an attractive dispersive component and a repulsive one for formation of a cavity in water. This allows us to identify the contributions to host-guest binding and to conclude that the binding process is driven by differential cavitation energies rather than dispersion interactions. The free energy required to create a cavity to accept the noble gas inside the cucurbit[5]uril is much lower than that to create a similarly sized cavity in bulk water. The recovery of the latter cavitation energy drives the overall process, which has implications for the refinement of gas-storage materials and the understanding of biological receptors.
Balasco N, Smaldone G, Ruggiero A, et al., 2018, Local structural motifs in proteins: Detection and characterization of fragments inserted in helices, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, Vol: 118, Pages: 1924-1930, ISSN: 0141-8130
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- Citations: 6
Patel JR, Xu Y, Capitini C, et al., 2018, Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 12, Pages: 273-277, ISSN: 1874-2718
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- Citations: 1
Gianni S, McCully ME, Malagrino F, et al., 2018, A Carboxylate to Amide Substitution That Switches Protein Folds, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, Vol: 57, Pages: 12795-12798, ISSN: 1433-7851
Balasco N, Barone D, Iaccarino E, et al., 2018, Intrinsic structural versatility of the highly conserved 412-423 epitope of the Hepatitis C Virus E2 protein, INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, Vol: 116, Pages: 620-632, ISSN: 0141-8130
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- Citations: 7
Capitini C, Patel JR, Natalello A, et al., 2018, Structural differences between toxic and nontoxic HypF-N oligomers, CHEMICAL COMMUNICATIONS, Vol: 54, Pages: 8637-8640, ISSN: 1359-7345
Fusco G, Sanz-Hernandez M, Ruggeri FS, et al., 2018, Molecular determinants of the interaction of EGCG with ordered and disordered proteins, BIOPOLYMERS, Vol: 109, ISSN: 0006-3525
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- Citations: 25
Romano M, Fusco G, Choudhury H, et al., 2018, Structural basis for natural product selection and export by bacterial ABC transporters, ACS Chemical Biology, Vol: 13, Pages: 1598-1609, ISSN: 1554-8929
Bacteria under stress produce ribosomally synthesized and post-translationally modified peptides (RiPPs) to target closely related species, such as the lasso peptide microcin J25 (MccJ25). These peptides are also toxic to the producing organisms that utilize dedicated ABC transporters to achieve self-immunity. MccJ25 is exported by the Escherichia coli ABC transporter McjD through a complex mechanism of recognition that has remained elusive. Here, we used biomolecular NMR to study this interaction and identified a region of the toxic peptide that is crucial to its recognition by the ABC transporter. Our study provides evidence that McjD is highly specific to MccJ25 and not to other RiPPs or antibiotics, unlike multidrug ABC transporters. Additionally, we show that MccJ25 is not exported by another natural product ABC transporter. Therefore, we propose that specific interactions between natural product ABC transporters and their substrate provides them with their high degree of specificity. Taken together, these findings suggest that ABC transporters might have acquired structural elements in their binding cavity to recognize and allow promiscuous export of a larger variety of compounds.
Lautenschlager J, Stephens AD, Fusco G, et al., 2018, C-terminal calcium binding of alpha-synuclein modulates synaptic vesicle interaction, Nature Communications, Vol: 9, Pages: 1-13, ISSN: 2041-1723
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.
Squeglia F, Ruggiero A, De Simone A, et al., 2018, A structural overview of mycobacterial adhesins: Key biomarkers for diagnostics and therapeutics, PROTEIN SCIENCE, Vol: 27, Pages: 369-380, ISSN: 0961-8368
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- Citations: 19
Fusco G, Sanz-Hernandez M, De Simone A, 2018, Order and disorder in the physiological membrane binding of α-synuclein, CURRENT OPINION IN STRUCTURAL BIOLOGY, Vol: 48, Pages: 49-57, ISSN: 0959-440X
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- Citations: 48
Fusco G, Chen SW, Williamson PTF, et al., 2017, Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers, Science, Vol: 358, Pages: 1440-1443, ISSN: 0036-8075
Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons.
Sanz-Hernandez M, De Simone A, 2017, The PROSECCO server for chemical shift predictions in ordered and disordered proteins, JOURNAL OF BIOMOLECULAR NMR, Vol: 69, Pages: 147-156, ISSN: 0925-2738
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- Citations: 8
Toto A, Bonetti D, De Simone A, et al., 2017, Understanding the mechanism of binding between Gab2 and the C terminal SH3 domain from Grb2., Oncotarget, Vol: 8, Pages: 82344-82351
Gab2 is a large disordered protein that regulates several cellular signalling pathways and is overexpressed in different forms of cancer. Because of its disordered nature, a detailed characterization of the mechanisms of recognition between Gab2 and its physiological partners is particularly difficult. Here we provide a detailed kinetic characterization of the binding reaction between Gab2 and the C-terminal SH3 domain of the growth factor receptor-bound protein 2 (Grb2). We demonstrate that Gab2 folds upon binding following an induced fit type mechanism, whereby recognition is characterized by the formation of an intermediate, in which Gab2 is primarily disordered. In this scenario, folding of Gab2 into the bound conformation occurs only after binding. However, an alanine scanning of the proline residues of Gab2 suggests that the intermediate contains some degree of native-like structure, which might play a role for the recognition event to take place. The results, which represent a fundamental step forward in the understanding of this functional protein-protein interaction, are discussed on the light of previous structural works on these proteins.
Heller GT, Aprilel FA, Bonomi M, et al., 2017, Sequence Specificity in the Entropy-Driven Binding of a Small Molecule and a Disordered Peptide, JOURNAL OF MOLECULAR BIOLOGY, Vol: 429, Pages: 2772-2779, ISSN: 0022-2836
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- Citations: 48
Toto A, Bonetti D, De Simone A, et al., 2017, Understanding the mechanism of binding between Gab2 and the C terminal SH3 domain from Grb2., Oncotarget
Gab2 is a large disordered protein that regulates several cellular signalling pathways and is overexpressed in different forms of cancer. Because of its disordered nature, a detailed characterization of the mechanisms of recognition between Gab2 and its physiological partners is particularly difficult. Here we provide a detailed kinetic characterization of the binding reaction between Gab2 and the C-terminal SH3 domain of the growth factor receptor-bound protein 2 (Grb2). We demonstrate that Gab2 folds upon binding following an induced fit type mechanism, whereby recognition is characterized by the formation of an intermediate, in which Gab2 is primarily disordered. In this scenario, folding of Gab2 into the bound conformation occurs only after binding. However, an alanine scanning of the proline residues of Gab2 suggests that the intermediate contains some degree of native-like structure, which might play a role for the recognition event to take place. The results, which represent a fundamental step forward in the understanding of this functional protein-protein interaction, are discussed on the light of previous structural works on these proteins.
Fusco G, Pape T, Stephens AD, et al., 2017, Structural basis of synaptic vesicle assembly promoted by alpha-synuclein (vol 7, 12563, 2016), NATURE COMMUNICATIONS, Vol: 8, ISSN: 2041-1723
Fusco G, Pape T, Stephens AD, et al., 2016, Structural basis of synaptic vesicle assembly promoted by α-synuclein, Nature Communications, Vol: 7, Pages: 1-12, ISSN: 2041-1723
α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson’s disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson’s disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this ‘double-anchor’ mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts.
Ruggiero A, Squeglia F, Romano M, et al., 2016, Structure and dynamics of the multi-domain resuscitation promoting factor RpfB from Mycobacterium tuberculosis, JOURNAL OF BIOMOLECULAR STRUCTURE & DYNAMICS, Vol: 35, Pages: 1322-1330, ISSN: 0739-1102
Fusco G, De Simone A, Arosio P, et al., 2016, Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity, Scientific Reports, Vol: 6, ISSN: 2045-2322
A detailed characterisation of the molecular determinants of membrane binding by α-synuclein (αS), a 140-residue protein whose aggregation is associated with Parkinson's disease, is of fundamental significance to clarify the manner in which the balance between functional and dysfunctional processes are regulated for this protein. Despite its biological relevance, the structural nature of the membrane-bound state αS remains elusive, in part because of the intrinsically dynamic nature of the protein and also because of the difficulties in studying this state in a physiologically relevant environment. In the present study we have used solid-state NMR and restrained MD simulations to refine structure and topology of the N-terminal region of αS bound to the surface of synaptic-like membranes. This region has fundamental importance in the binding mechanism of αS as it acts as to anchor the protein to lipid bilayers. The results enabled the identification of the key elements for the biological properties of αS in its membrane-bound state.
Sanfelice D, Sanz-Hernandez M, De Simone A, et al., 2016, Towards Understanding the Molecular Bases of Stretch Activation: A Structural comparison of the Two Troponin C Isoforms of Lethocerus., Journal of Biological Chemistry, Vol: 291, Pages: 16090-16099, ISSN: 1083-351X
Muscles are usually activated by calcium binding to the calcium-sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle (IFM) activation is also provided by mechanical stress. Little is known about the molecular bases of this calcium-independent activation. In Lethocerus, a giant water bug often used as a model system thanks to the big size of its muscle fibers, there are two troponin-C isoforms, named F1 and F2, which seem to have very distinct roles and mechanical properties. It has been suggested that this behaviour can be explained either by differences in structural features, or by differences in the interactions with other proteins. Here, we have compared the structural and dynamic properties of the two proteins and shown that the two proteins have intrinsically different behaviours. We also mapped the interactions of the F2 isoform with peptides spanning the sequence of its natural partner, troponin I. Our data allowed us to build a model of the troponin complex and may eventually help understanding the specialised function of the F1 isoform and the molecular mechanism of stretch activation.
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