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Biomedical subjects

Maurizio Paci

Publications and source records attributed to Maurizio Paci.

At least 19 recordsLinked to original sources
PubMed2006

Metal-binding and nuclease activity of an antimicrobial peptide analogue of the salivary histatin 5.

The salivary antimicrobial peptide histatin 5 is characterized by its cationic nature, structural flexibility, and the presence of two metal-binding sites (the ATCUN motif and a Zn-binding motif). These properties make this peptide a good model for the design of new drugs of low molecular weight. In this work, we have synthesized and studied a new peptide, an analogue of the histatin 5 named ATCUN-C16, which contains both metal-binding centers. The results show that our 20-residue-derived peptide preserves anticandidal activity and exhibits a higher propensity to assume a stable conformation in a hydrophobic environment than do histatin 5 and the C16 peptide that contains the 16 residues of the C-terminal part of histatin 5, although overall our peptide remains a flexible molecule. ACTUN-C16 was found to bind DNA in a gel retardation assay and to have a nuclease activity in the presence of copper and zinc ions and ascorbate. Its nuclease activity can be attributed to the synergistic action of oxidative and hydrolytic activities due to the Cu-ATCUN complex and to the zinc ion coordination, respectively. The results show a new property of this family of salivary peptides and suggest a novel use of this peptide as a small nuclease and biotechnological tool.

Amino Acid Sequence↗
PubMed2006

Structural basis for the interaction of the myosin light chain Mlc1p with the myosin V Myo2p IQ motifs.

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.

Amino Acid Motifs↗
PubMed2006

Structural and thermodynamic basis for the enhanced transcriptional control by the human papillomavirus strain-16 E2 protein.

Strain 16 of the human papillomavirus is responsible for the largest number of cases of cervical cancers linked to this virus, and the E2 protein is the transcriptional regulator of all viral genes. We present the first structure for the DNA binding domain of HPV16 E2 bound to DNA, and in particular, to a natural cognate sequence. The NMR structure of the protein backbone reveals that the overall conformation remains virtually unchanged, and chemical shift analysis of the protein bound to a shorter DNA duplex uncovered a contact out of the minimal E2 DNA binding site, made by lysine 349. This contact was confirmed by titration calorimetry and mutagenesis, with a contribution of 1.0 kcal mol(-)(1) to binding energy. HPV16 E2 has the highest DNA binding affinity and exerts a strict transcriptional control, translated into the repression of the E6 and E7 oncogenes. These novel features provide the structural and thermodynamic basis for this tight transcriptional control, the loss of which correlates with carcinogenesis.

Amino Acid Sequence↗
PubMed2006

On the effect of a point mutation on the reactivity of CuZn superoxide dismutase: a theoretical study.

In this paper, we investigate the effects of a point mutation on the enzymatic activity of copper-zinc superoxide dismutase, which we recently studied in detail by means of a theoretical-computational procedure. Comparison of the reactivity of the initial catalytic steps in this mutant (G93A mutation far from the active site) with our previous data, reveals the beautiful mechanical-dynamical architecture of the enzyme, altered by such an apparently irrelevant mutation. Finally, our results suggest a possible atomic-molecular-based explanation for the mutant-pathology correlation, in line with the most recent experimental data.

Electromagnetic Fields↗
PubMed2006

HACACO revisited: residual dipolar coupling measurements and resonance assignments in proteins.

The revisited version of the HACACO experiment here presented, is more robust and straightforward to implement and continues to be, to a greater extent, a convenient tool for protein backbone resonance assignment. Additionally, it turns out to be a sensitive and accurate method to measure C(alpha)-H(alpha) residual dipolar couplings (RDCs). The performance of our new pulse scheme for measurement of RDCs was tested on two proteins with different secondary structures: one characterized by a high beta-sheet content, the second dominated by the presence of alpha-helices. In both examples the new method provided significantly more accurate data, compared to all previously published 3D techniques.

Bacterial Proteins↗
PubMed2006

Cloning, expression, and preliminary structural characterization of RTN-1C.

Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.

Circular Dichroism↗
PubMed2006

Molecular basis for phosphorylation-dependent, PEST-mediated protein turnover.

Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and alpha helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.

Amino Acid Motifs↗
PubMed2006

The active essential CFNS3d protein complex.

The NS2B-NS3 protease complex is essential for the replication of dengue virus, which is the etiologic agent of dengue and hemorrhagic fevers, diseases that are a burden for the tropical and subtropical areas of the world. The active form of the NS3 protease linked to the 40 residues of the NS2B cofactor shows highly flexible and disordered region(s) that are responsible for its high propensity to aggregate at the concentrations necessary for NMR spectroscopy studies or for crystallization. Limited proteolysis of this active form of the protease enabled us to obtain a folded and new essential form of the NS2B-NS3 protease complex. We found that the region from residues D50 to E80 of NS2B interacts directly and strongly with the NS3 protease domain. The proteolytic activity of the noncovalently binding complex was determined by a rapid and continuous fluorescence resonance energy transfer activity assay using a depsipeptide substrate. The new protein-cofactor complex obtained, encompassing the NS2B fragment (D50-E80) and the NS3 protease, shows proteolytic activity. The (1)H-(15)N-heteronuclear single quantum coherence spectrum of the isotopically enriched protein complex shows good cross-peak dispersion; this is indicative of a stable folded state. Our results significantly complement the X-ray structure of the NS2B-NS3pro complex published recently. Moreover, these results open the way to performing direct structural and interaction studies in solution on a new active NS2B-NS3pro complex with libraries of substrates and inhibitors in order to identify new drugs that prevent viral polyprotein processing.

Amino Acid Sequence↗
PubMed2005

Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family.

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Amino Acid Motifs↗
PubMed2004

The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain.

Sulfurtransferase are enzymes involved in the formation, conversion and transport of compounds containing sulfane-sulfur atoms. Although the three-dimensional structure of the rhodanese from the nitrogen-fixing bacterium Azotobacter vinelandii is known, the role of its two domains in the protein conformational stability is still obscure. We have evaluated the susceptibility to proteolytic degradation of the two domains of the enzyme. The two domains show different resistance to the endoproteinases and, in particular, the N-terminal domain shows to be more stable to digestion during time than the C-terminal one. Cloning and overexpression of the N-terminal domain of the protein was performed to better understand its functional and structural role. The recombinant N-terminal domain of rhodanese A. vinelandii is soluble in water solution and the spectroscopic studies by circular dichroism and heteronuclear NMR spectroscopy indicate a stable fold of the protein with the expected alpha/beta topology. The results indicate that this N-terminal domain has already got all the elements necessary for an C-terminal domain independent folding. Its solution structure by NMR, actually under course, will be a valid contribution to understand the role of this domain in the folding process of the sulfurtransferase.

Amino Acid Sequence↗
PubMed2004

The effect of an ionic detergent on the natively unfolded beta-dystroglycan ectodomain and on its interaction with alpha-dystroglycan.

Dystroglycan (DG) is an adhesion complex, expressed in a wide variety of tissues, formed by an extracellular and a transmembrane subunit, alpha-DG and beta-DG, respectively, interacting noncovalently. Recently, we have shown that the recombinant ectodomain of beta-DG, beta-DG(654-750), behaves as a natively unfolded protein, as it is able to bind the C-terminal domain of alpha-DG, while not displaying a defined structural organization. We monitored the effect of a commonly used denaturing agent, the anionic detergent sodium dodecylsulphate (SDS), on beta-DG(654-750) using a number of biophysical techniques. Very low concentrations of SDS (< or =2 mM) affect both tryptophan fluorescence and circular dichroism of beta-DG, and significantly perturb the interaction with the alpha-DG subunit as shown by solid-phase binding assays and fluorescence titrations in solution. This result confirms, as recently proposed for natively unfolded proteins, that beta-DG(654-750) exists in a native state, which is crucial to fulfill its biological function. Two-dimensional NMR analysis shows that SDS does not induce any evident conformational rearrangement within the ectodomain of beta-DG. Its first 70 amino acids, which show a lower degree of mobility, interact with the detergent, but this does not change the amount of secondary structure, whereas the highly flexible and mobile C-terminal region of beta-DG(654-750) remains largely unaffected, even at a very high SDS concentration (up to 50 mM). Our data indicate that SDS can be used as a useful tool for investigating natively unfolded proteins, and confirm that the beta-DG ectodomain is an interesting model system.

Amino Acid Sequence↗
PubMed2004

Solution structure of the cyclic peptide contryphan-Vn, a Ca2+-dependent K+ channel modulator.

The solution structure of contryphan-Vn, a cyclic peptide with a double cysteine S-S bridge and containing a D-tryptophan extracted from the venom of the cone snail Conus ventricosus, has been determined by NMR spectroscopy using a variety of homonuclear and heteronuclear NMR methods and restrained molecular dynamics simulations. The main conformational features of backbone contryphan-Vn are a type IV beta-turn from Gly 1 to Lys 6 and a type I beta-turn from Lys 6 to Cys 9. As already found in other contryphans, one of the two prolines--the Pro4--is mainly in the cis conformation while Pro7 is trans. A small hydrophobic region probably partly shielded from solvent constituted from the close proximity of side chains of Pro7 and Trp8 was observed together with a persistent salt bridge between Asp2 and Lys6, which has been revealed by the diagnostic observation of specific nuclear Overhauser effects. The salt bridge was used as a restraint in the molecular dynamics in vacuum but without inserting explicit electrostatic contribution in the calculations. The backbone of the unique conformational family found of contryphan-Vn superimposes well with those of contryphan-Sm and contryphan-R. This result indicates that the contryphan structural motif represents a robust and conserved molecular scaffold whose main structural determinants are the size of the intercysteine loop and the presence and location in the sequence of the D-Trp and the two Pro residues.

Animals↗
PubMed2004

Conformational fluctuations and electronic properties in myoglobin.

In this article we use the recently developed perturbed matrix method (PMM) to investigate the effect of conformational fluctuations on the electronic properties of heme in Myoglobin. This widely studied biomolecule has been chosen as a benchmark for evaluating the accuracy of PMM in a large and complex system. Using a long, 80-ns, molecular dynamics simulation and unperturbed Configuration Interaction (CISD) calculations in PMM, we reproduced the main spectroscopic features of deoxy-Myoglobin. Moreover, in line with our previous results on a photosensitive protein, this study reveals a clear dynamical coupling between electronic properties and conformational fluctuations, suggesting that this correlation could be a general feature of proteins.

Algorithms↗
PubMed2003

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

Amino Acid Sequence↗