Biomedical subjects

M A Castiglione Morelli

Publications and source records attributed to M A Castiglione Morelli.

15 recordsLinked to original sources

PubMed2001

Kinase recognition by calmodulin: modeling the interaction with the autoinhibitory region of human cardiac titin kinase.

Calmodulin (CaM)-protein interactions are usually described by studying complexes between synthetic targets of ca 25 amino acids and CaM. To understand the relevance of contacts outside the protein-binding region, we investigated the complex between recombinant human CaM (hCaM) and P7, a 38-residue peptide corresponding to the autoinhibitory domain of human cardiac titin kinase (hTK). To expedite the structure determination of hCaM-P7 we relied upon the high degree of similarity with other CaM-kinase peptide complexes. By using a combined homonuclear NMR spectroscopy and molecular modeling approach, we verified for the bound hCaM similar trends in chemical shifts as well as conservation of NOE patterns, which taken together imply the conservation of CaM secondary structure. P7 was anchored to the protein with 52 experimental intermolecular contacts. The hCaM-P7 structure is very similar to known CaM complexes, but the presence of NOE contacts outside the binding cavity appears to be novel. Comparison with the hTK crystal structure indicates that the P7 charged residues all correspond to accessible side-chains, while the putative anchoring hydrophobic side-chains are partially buried. To test this finding, we also modeled the early steps of the complex formation between Ca(2+)-loaded hCaM and hTK. The calculated trajectories strongly suggest the existence of an "electrostatic funnel", driving the long-range recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residues remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca(2+)/hCaM, favoring the intrasteric regulation of the kinase. Furthermore, our structure, the first of CaM bound to a peptide derived from a kinase whose three-dimensional structure is known, suggests that special care is needed in the choice of template peptides to model protein-protein interactions.

Amino Acid Sequence↗

PubMed1999

Novel RNA-binding motif: the KH module.

The KH motif has recently been identified in single or multiple copies in a number of RNA associated proteins. Here we review the current knowledge accumulated about the sequence, structure, and functions of the KH. The multidomain architecture of most of the KH-containing proteins inspired an approach based on the production of peptides spanning the sequence of an isolated KH motif. Correct identification of the minimal length necessary for producing a folded peptide has had a number of important consequences for interpreting functional data. The presence of the KH motifs in fmr1, the protein responsible for the fragile X syndrome, and their possible role in the fmr1 functions are also discussed.

Amino Acid Sequence↗

PubMed1999

Conformational flexibility in calcitonin: the dynamic properties of human and salmon calcitonin in solution.

We have studied the dynamic properties of human (h) and salmon (s) calcitonin (CT) in solution. For both hormones, distance geometry in torsion-angle space has been used to generate three-dimensional structures consistent with NMR data obtained in sodium dodecyl sulfate micelles. For sCT and hCT we used, respectively, 356 and 275 interproton distances together with hydrogen-bonds as restraints. To better characterize their flexibility and dynamic properties two fully unrestrained 1100-ps molecular dynamics (MD) simulations in methanol were performed on the lowest-energy structures of both hormones. Statistical analyses of average geometric parameters and of their fluctuations performed in the last 1000 ps of the MD run show typical helical values for residues 9-19 of sCT during the whole trajectory. For hCT a shorter helix was observed involving residues 13-21, with a constant helical region in the range 13-19. Angular order parameters S(phi) and S(psi) indicate that hCT exhibits a higher flexibility, distributed along the whole chain, including the helix, while the only flexible amino acid residues in sCT connect three well-defined domains. Finally, our study shows that simulated annealing in torsion-angle space can efficiently be extended to NMR-based three-dimensional structure calculations of helical polypeptides. Furthermore, provided that a sufficient number of NMR restraints describes the system, the method allows the detection of equilibria in solution. This identification occurs through the generation of 'spurious' high-energy structures, which, for right-handed alpha-helices, are likely to be represented by left-handed alpha-helices.

Amino Acid Sequence↗

PubMed1998

Solution structure of human calcitonin in membrane-mimetic environment: the role of the amphipathic helix.

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHNalpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic alpha-helix from Leu9 to Phel6, followed by a type-I beta-turn between residues Phel6 and Phel9. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure-activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed.

Amino Acid Sequence↗

PubMed1997

A new triple-stranded alpha-helical bundle in solution: the assembling of the cytosolic tail of MHC-associated invariant chain.

BACKGROUND: The invariant chain (li) is a transmembrane protein that associates with the major histocompatibility complex class II (MHC II) molecules in the endoplasmic reticulum. The cytosolic tail of li contains two leucine-based sorting motifs and is involved in sorting the MHC II molecules to the endosomal pathway where the peptide antigen is bound. This region of li also contributes to phenotypical changes in cells, such as the formation of large endocytic structures. RESULTS: We report here the three-dimensional structure of a 27 amino acid peptide corresponding to the cytosolic tail of li. The structure was determined by nuclear magnetic resonance (NMR) spectroscopy using a computational strategy. At high concentration, this structure reveals a new triple-stranded alpha-helical bundle in which the helices, two parallel and one antiparallel, are almost coplanar. Trimerization is mediated by electrostatic interactions intercalated by three hydrophobic layers. CONCLUSIONS: The new trimer fold, the first to be identified by NMR data alone, can be used to improve understanding of protein-protein interactions and to model multiple-helical transmembrane proteins and receptors. We suggest that interactions of the li cytosolic tails may form part of a mechanism that could cause the endosomal retention and enlarged endosomes induced by li.

Amino Acid Sequence↗

PubMed1997

Structure-activity relationships for some elastin-derived peptide chemoattractants.

In an attempt to explore the relationships between conformation of chemotactic peptides related to elastin and their biological activity we have studied five peptides: VGVAPG, VGVPG, VGAPG, GVAPG and GGVPG in solvents of different polarities which may mimic the environmental conditions at the receptor site. CD and NMR studies showed that GVAPG has no preference for structured conformations, while the other peptides may assume folded conformations in organic solvents. All these peptides but GGVPG showed chemotactic activity for monocytes. The chemotactic activity of VGVPG, VGAPG and VGVAPG was inhibited by lactose, while chemotaxis of peptide GVAPG was insensitive to lactose, suggesting the existence of different chemotactic receptors.

Chemotactic Factors↗

PubMed1996

Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome.

The KH module is a sequence motif found in a number of proteins that are known to be in close association with RNA. Experimental evidence suggests a direct involvement of KH in RNA binding. The human FMR1 protein, which has two KH domains, is associated with fragile X syndrome, the most common inherited cause of mental retardation. Here we present the three-dimensional solution structure of the KH module. The domain consists of a stable beta alpha alpha beta beta alpha fold. On the basis of our results, we suggest a potential surface for RNA binding centered on the loop between the first two helices. Substitution of a well-conserved hydrophobic residue located on the second helix destroys the KH fold; a mutation of this position in FMR1 leads to an aggravated fragile X phenotype.

Asparagine↗

PubMed1996

Correlation between conformational and binding properties of nebulin repeats.

Nebulin, a large protein (600 to 800 kDa) located in the thin filament of striated vertebrate muscle, is assumed to bind and stabilise F-actin. Complete sequence determination of human nebulin has only recently been accomplished showing a uniform modular structure along the whole length of the molecule. Up to 97% of the sequence is assembled from repeats of a sequence motif 35 amino acid residues long. This architecture suggests that a structural and functional understanding of such a large molecule may be possible by characterising single repeats and reconstructing from them the behaviour of the whole molecule. In the present study, we extend and generalise to the whole molecule previous work carried out on single repeats from a limited region of nebulin. Knowledge of the complete sequence allowed extensive analysis of the single repeats revealing a progressive N to C-terminal divergence that is mirrored by an increase of the alpha-helix propensity. A number of synthetic peptides spanning the sequences of selected repeats were obtained and their conformational and binding properties studied in detail. All the peptides showed a tendency to fold as transient helices in aqueous solution with helix content as observed by CD and NMR studies in excellent agreement with predictions. A higher helical tendency of repeats near the C terminus was observed. Analysis of the influence of charged media as well as trifluoroethanol on the folding of single repeats strongly suggested that the mechanism by which the nebulin alpha-helix is stabilised is mostly electrostatic. Peptides with higher helical content also showed a higher binding affinity to F-actin. Considerably varying effects were observed for the peptides on F-actin viscosity and polymerisation. We discuss the divergence in sequence and helical tendency and its correlation to the functional data with regard to their significance for the assembly of the thin filament during myogenesis.

Actins↗

PubMed1995

A calmodulin-binding sequence in the C-terminus of human cardiac titin kinase.

The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C-terminus which bears similarity to the substrate. A calmodulin-binding sequence has also been identified in the C-terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin-binding site in the titin kinase C-terminal sequence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C-terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interaction of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt alpha-helical conformations in solution. Helicity increases upon binding of calmodulin in a calcium-dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin-binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.

Amino Acid Sequence↗

PubMed1992

Dynamic properties of salmon calcitonin bound to sodium dodecyl sulfate micelles: a restrained molecular dynamics study from NMR data.

We have investigated the conformational behaviour of salmon calcitonin bound to sodium dodecyl sulfate micelles by means of restrained molecular dynamics simulations with both 'static' and time-averaged NMR distance restraints. A more realistic picture of the inherent flexibility of the hormone is obtained when using time averaging. With this approach, long-range NOEs are interpreted better by considering a dynamical exchange among different conformations.

Amino Acid Sequence↗

PubMed1992

The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?

LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.

DNA-Binding Proteins↗

PubMed1991

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations.

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.

Amino Acid Sequence↗

PubMed1991

1H resonance assignment and secondary structure determination of the dimerization domain of transcription factor LFB1.

We have started the structure determination of the dimerization domain of LFB1 in solution by nuclear magnetic resonance in order to elucidate the way that the LFB1 protein dimerizes and then interacts with DNA. A 32 amino acid peptide was synthesized, and full assignment of the NMR resonances in acidic solution was achieved. The secondary structure determination is presented here. Three structurally distinct regions can be distinguished. The N-terminal region from residues 1 to 6 is extended. Two helical regions span from residues 7 to 18 and from 23 to 32. The absence of dipolar effects involving residues more than four positions apart in the sequence excludes the possibilities both of a four-helix bundle formed by two hairpins and of an antiparallel dimer; the domain must therefore be arranged as a parallel dimer formed by kinked monomers. This structural solution presents important differences from the leucine zipper-type structure observed in other transcriptional activators. Although further studies are still necessary to determine the 3D structure of the peptide, we can exclude the possibility of a coiled-coil structure.

Amino Acid Sequence↗

PubMed1991

Conformational study of cyclolinopeptide A. A distance geometry and molecular dynamics approach.

The conformation of cyclolinopeptide A, c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of distance geometry calculations and molecular dynamics simulations. The starting points for all the calculations were an X-ray structure and other structures obtained from distance geometry calculations based on NMR data. Restrained and unrestrained molecular dynamics simulations are reported in vacuo and in CCl4. Structural and dynamic properties are investigated and compared with those experimentally determined. The conformation obtained from the MD simulations which best reproduces the NMR parameters is at the same time one of the most stable ones and is also fairly similar to the crystal structure. An explanation for the occurrence of multiple conformations in solution at room temperature is given.

Amino Acid Sequence↗

PubMed1990

Solution structure of the DNA-binding domain of the yeast transcriptional activator protein GCN4.

The solution structure of an active synthetic peptide containing both the leucine zipper and the adjacent basic domain of the yeast transcription factor GCN4 (residues 220-280) was determined by NMR. The two domains show structurally distinct behaviours. In the absence of DNA, the basic domain is, although very flexible, structured and fluctuating around a helical conformation. The leucine zipper region forms a long, uninterrupted helix. From a suitable set of NMR distances the three-dimensional structure of the leucine zipper monomeric sub-domain was calculated by distance geometry algorithms. The structure of the symmetrical parallel dimer was obtained by model building using the NMR information. A smaller peptide with the sequence of the isolated basic region (residues 1-35 of the 61 residue peptide) was also synthesized. Circular dichroism studies showed 30-40% helicity. A flexible helix spans the region between residues 8 and 21. The comparison of our results with suggested models is discussed in detail.

Algorithms↗