Biomedical subjects

Roberta Spadaccini

Publications and source records attributed to Roberta Spadaccini.

10 recordsLinked to original sources

PubMed2006

Structure and functional analysis of the MYND domain.

The MYND domain (named after myeloid translocation protein 8, Nervy, and DEAF-1) is a conserved zinc binding domain. It is defined by seven conserved cysteine residues and a single histidine residue that are arranged in a C4-C2HC consensus. MYND domains exist in a large number of proteins that play important roles in development or are associated with cancers and have been shown to mediate protein-protein interactions, mainly in the context of transcriptional regulation. We have determined the three-dimensional structure of the MYND domain from human deformed epidermal autoregulatory factor-1 (DEAF-1). The structure reveals a novel zinc binding fold, in which the C4-C2HC motif forms two sequential zinc binding sites. The first and second zinc binding modules comprise a small beta hairpin and two short alpha helices, respectively. The sequential topology of the two zinc binding sites is distinct from the cross-brace PHD and RING finger folds but has some resemblance to LIM domains. The structure reveals that the MYND domain is a novel member of the treble-clef family of zinc binding domains. The MYND domains of BS69 and BOP bind ligands comprising a PXLXP peptide motif. On the basis of the solution structure of the DEAF-1 MYND domain we calculated a homology model of the MYND domain of the BS69 tumor suppressor. A mutational analysis of the BS69 MYND domain indicates that positively charged residues located on one face of its MYND domain are crucial for the molecular interactions of BS69. Different binding specificities of MYND domains may depend on distinct surface charge distributions.

Amino Acid Sequence↗

PubMed2006

Toward the understanding of MNEI sweetness from hydration map surfaces.

The binding mechanism of sweet proteins to their receptor, a G-protein-coupled receptor, is not supported by direct structural information. In principle, the key groups responsible for biological activity (glucophores) can be localized on a small structural unit (sweet finger) or spread on a larger surface area. A recently proposed model, called "wedge model", implies a large surface of interaction with the receptor. To explore this model in greater detail, it is necessary to examine the physicochemical features of the surfaces of sweet proteins, since their interaction with the receptor, with respect to that of small sweeteners, is more dependent on general physicochemical properties of the interface, such as electrostatic potential and hydration. In this study, we performed exhaustive molecular dynamics simulations in explicit water of the sweet protein MNEI and of its structural mutant G-16A, whose sweetness is one order of magnitude lower than that of MNEI. Solvent density and self-diffusion calculated from molecular dynamics simulations suggest a likely area of interaction delimited by four stretches arranged as a tetrahedron whose shape is complementary to that of a cavity on the surface of the receptor, in agreement with the wedge model. The suggested area of interaction is amazingly consistent with known mutagenesis data. In addition, the asymmetric hydration of the only helix in both proteins hints at a specific role for this secondary structure element in orienting the protein during the binding process.

Computer Simulation↗

PubMed2006

Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing.

The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.

Amino Acid Sequence↗

PubMed2005

Structural and functional studies of vertebrate metallothioneins: cross-talk between domains in the absence of physical contact.

In previous studies, we showed that the chemical and dynamic properties of fish and mouse MTs (metallothioneins) present a number of distinctive differences linked to their primary structures, and that phylogenetic relationships of mammal and fish MTs correlate with their three-dimensional structures. The different behaviours of MTs may also be linked to the interaction between their two domains. In the present study, we have compared the physicochemical properties of the isolated recombinant domains constituting Notothenia coriiceps and mouse MTs, and compared them with those of the corresponding whole MTs. NMR spectra of the separated domains of N. coriiceps are almost superimposable on those of the parent MT, suggesting an apparent lack of interaction between the two domains in the protein. However, certain dynamic and physicochemical features of the isolated domains are unlike those of the whole protein. In particular, the temperature-induced changes in the chiroptical properties, thiol reactivity of the Zn-MT domains and the Zn2+/Cd2+ rate of exchange are different for the two domains and with respect to the whole protein. Taken together, these results provide a strong argument in favour of the interaction of the two domains in the MT molecule, in spite of the elusive evidence provided by the structural analyses.

Animals↗

PubMed2003

The swapping of terminal arms in ribonucleases: comparison of the solution structure of monomeric bovine seminal and pancreatic ribonucleases.

Bovine seminal ribonuclease (BS-RNase), the only dimeric protein among the pancreatic-like ribonucleases, is endowed with special structural features and with biological functions beyond enzymatic activity. In solution, the protein exists as an equilibrium mixture of two forms, with or without exchange (or swapping) of the N-terminal arms. After selective reduction and alkylation of the two intrachain disulfide bridges, the dimeric protein can be transformed into a monomeric derivative that has a ribonuclease activity higher than that of the parent dimeric protein but is devoid of the special biological functions. A detailed investigation of the structural features of this protein in solution, in comparison with those of other monomeric ribonucleases, may help unveil the structural details which induce swapping of the N-terminal arms of BS-RNase. The solution structure of the recombinant monomeric form of BS-RNase, as determined by 3D heteronuclear NMR, shows close similarity with that of bovine pancreatic ribonuclease (RNase A) in all regions characterized by regular elements of secondary structure. However, significant differences are present in the flexible regions, which could account for the different behavior of the two proteins. To characterize in detail these regions, we have measured H/D exchange rate constants, temperature coefficients and heteronuclear NOEs of backbone amides for both RNase A and monomeric BS-RNase. The results indicate a large difference in the backbone flexibility of the hinge peptide segment 16-22 of the two proteins, which could provide the molecular basis to explain the ability of BS-RNase subunits to swap their N-terminal arms.

Animals↗

PubMed2003

The mechanism of interaction of sweet proteins with the T1R2-T1R3 receptor: evidence from the solution structure of G16A-MNEI.

The mechanism by which sweet proteins elicit a response on the T1R2-T1R3 sweet taste receptor is still mostly unknown but has been so far related to the presence of "sweet fingers" on the protein surface able to interact with the same mechanism as that of low molecular mass sweeteners. In the search for the identification of sweet fingers, we have solved the solution structure of G16A MNEI, a structural mutant that shows a reduction of one order of magnitude in sweetness with respect to its parent protein, MNEI, a single-chain monellin. Comparison of the structures of wild-type monellin and its G16A mutant shows that the mutation does not affect the structure of potential glucophores but produces a distortion of the surface owing to the partial relative displacement of elements of secondary structure. These results show conclusively that sweet proteins do not possess a sweet finger and strongly support the hypothesis that the mechanism of interaction of sweet-tasting proteins with the recently identified T1R2-T1R3 GPC receptor is different from that of low molecular mass sweeteners.

Binding Sites↗

PubMed2003

Phylogenetic divergence of fish and mammalian metallothionein: relationships with structural diversification and organismal temperature.

Metallothioneins (MTs) are nonenzymatic low molecular weight proteins, that play an important role in the homeostasis and detoxification of heavy metals in a large variety of organisms. These proteins are endowed with striking features, including an unusual amino acid composition characterized by the presence of 20 cysteines out of a total of 60 residues and absence of secondary structure elements. It is generally accepted that MTs underwent few modifications during evolution because of these structural and functional constraints. Such a conclusion is founded on the studies carried out mostly on MTs of mammalian origin. For such a reason, we have decided to compare the MTs of homeothermic and poikilothermic organisms, such as mammals and fish, with the specific aim to put in relation phylogenetic divergence and structural/functional adaptation to temperature. We have included in our analysis also Antarctic Notothenioids, a fish group characterized by genetic isolation and cold-adaptation to a particular harsh environment. We have determined the average hydropathic index of ancestral MT sequences and used them to infer the temperatures of the environment housing the hypothetical ancestor organisms. Finally, we have derived phylogenetic relationships of MT molecules from the pairwise comparison of their three-dimensional structures.

Adaptation, Physiological↗

PubMed2003

Solution structure of MT_nc, a novel metallothionein from the Antarctic fish Notothenia coriiceps.

The structure of [113Cd(7)]-metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps, the first three-dimensional structure of a fish metallothionein, was determined by homonuclear 1H NMR experiments and heteronuclear [1H, 113Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal beta domain with 9 cysteines and 3 metal ions and a carboxy-terminal alpha-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the alpha domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the alpha domain and, in turn, to a different charge distribution with respect to that observed in mammalian metallothioneins.

Amino Acid Sequence↗

PubMed2002

The SH3 domain of nebulin binds selectively to type II peptides: theoretical prediction and experimental validation.

Nebulin, a giant modular protein from muscle, is thought to act as a molecular ruler in sarcomere assembly. The C terminus of nebulin, located in the sarcomere Z-disk, comprises an SH3 domain, a module well known for its role in protein/protein interactions. SH3 domains are known to recognize proline-rich ligands, which have been classified as type I or type II, depending on their relative orientation with respect to the SH3 domain in the complex formed. Type I ligands are bound with their N terminus at the RT loop of the SH3 domain, while type II ligands are bound with their C terminus at the RT loop. Many SH3 domains can bind peptides of either class. Despite the potential importance of the SH3 domain for the function of nebulin as an integral part of a complex network of interactions, no in vivo partner has been identified so far. We have adopted an integrated approach, which combines bioinformatic tools with experimental validation to identify possible partners of nebulin SH3. Using the program SPOT, we performed an exhaustive screening of the muscle sequence databases. This search identified a number of potential nebulin SH3 partners, which were then tested experimentally for their binding affinity. Synthetic peptides were studied by both fluorescence and NMR spectroscopy. Our results show that nebulin SH3 domain binds selectively to type II peptides. The affinity for a type II peptide, 12 residues long, spanning the sequence of a stretch of titin known to colocalise with nebulin in the Z-disk is in the submicromolar range (0.7 microM). This affinity is among the highest found for SH3/peptide complexes, suggesting that the identified stretch could have significance in vivo. The strategy outlined here is of more general applicability and may provide a valuable tool to identify potential partners of SH3 domains and of other peptide-binding modules.

Amino Acid Sequence↗

PubMed2002

Solution structure of nociceptin peptides.

Peptides embedded in the sequence of pre-pro-nociceptin, i.e. nociceptin, nocistatin and orphanin FQ2, have shed light on the complexity of the mechanisms involving the peptide hormones related to pain and have opened up new perspectives for the clinical treatment of pain. The design of new ligands with high selectivity and bioavailability, in particular for ORL1, is important both for the elucidation and control of the physiological role of the receptor and for their therapeutic importance. The failure to obtain agonists and antagonists when using, for nociceptin, the same substitutions that are successful for opioids, and the conformational flexibility of them all, justify systematic efforts to study the solution conformation under conditions as close as possible to their natural environment. Structural studies of linear peptides in solution are hampered by their high flexibility. A direct structural study of the complex between a peptide and its receptor would overcome this difficulty, but such a study is not easy since opioid receptors are membrane proteins. Thus, conformational studies of lead peptides in solution are still important for drug design. This review deals with conformational studies of natural pre-nociceptin peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yielded a completely reliable bioactive conformation, but the global conformation of the peptides in biomimetic environments can shed light on their interaction with receptors.

Animals↗