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Gabriele Cavallaro

Publications and source records attributed to Gabriele Cavallaro.

10 recordsLinked to original sources
PubMed2006

Structural basis for the observed differential magnetic anisotropic tensorial values in calcium binding proteins.

Lanthanide ions (Ln(3+)), which have ionic radii similar to those of Ca(2+), can displace the latter in a calcium binding protein, without affecting its tertiary structure. The paramagnetic Ln(3+) possesses large anisotropic magnetic susceptibilities and produce pseudocontact shifts (PCSs), which have r(-3) dependence. The PCS can be seen for spins as far as 45 A from the paramagnetic ion. They aid in structure refinement of proteins by providing long-range distance constraints. Besides, they can be used to determine the interdomain orientation in multidomain proteins. This is particularly important in the context of a calcium binding protein from Entamoeba histolytica (EhCaBP), which consists of two globular domains connected by a flexible linker region containing 8 residues. As a first step to obtain the interdomain orientation in EhCaBP, a suite of 2D and 3D heteronuclear experiments were recorded on EhCaBP by displacing calcium with Ce(3+), Ho(3+), Er(3+), Tm(3+), Dy(3+), and Yb(3+) ions in separate experiments, and the PCS of (1)H(N) and (15)N spins were measured. Such data have been used in the refinement of the individual domain structures of the protein in parallel with the calculation of the respective magnetic anisotropy tensorial values, which differ substantially (2.1-2.8 times) from what is found in other Ca(2+) binding loops. This study provides a structural basis for such variations in the magnetic anisotropy tensorial values.

Animals↗
PubMed2006

Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal.

Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha3) and zinc (alpha5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.

Allosteric Regulation↗
PubMed2005

A structural model for the adduct between cytochrome c and cytochrome c oxidase.

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein-protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc (1) to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc (1)-cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space.

Cytochromes c↗
PubMed2004

Paramagnetism-based restraints for Xplor-NIH.

Modules that use paramagnetism-based NMR restraints have been developed and integrated in the well known program for solution structure determination Xplor-NIH; the complete set of such modules is called PARArestraints for Xplor-NIH. Paramagnetism-based restraints are paramagnetic relaxation enhancements, pseudocontact shifts, residual dipolar couplings due to metal and overall magnetic anisotropy, and cross correlation between Curie relaxation and nuclear-nuclear dipolar relaxation. The complete program has been tested by back-calculating NOEs and paramagnetism-based restraints from the X-ray structure of cytochrome c (553) from B. pasteurii. Furthermore, the same experimental restraints previously used to determine the solution structure of cytochrome c (553) itself, of cytochrome b (5), and of calbindin D(9k) with the program PARAMAGNETIC DYANA, have been used for structure calculations by using PARArestraints for Xplor-NIH. The agreement between the two programs is quite satisfactory and validates both protocols.

Calbindins↗
PubMed2003

A use of Ramachandran potentials in protein solution structure determinations.

A strategy is developed to use database-derived phi-psi constraints during simulated annealing procedures for protein solution structure determination in order to improve the Ramachandran plot statistics, while maintaining the agreement with the experimental constraints as the sole criterion for the selection of the family. The procedure, fully automated, consists of two consecutive simulated annealing runs. In the first run, the database-derived phi-psi constraints are enforced for all amino acids (but prolines and glycines). A family of structures is then selected on the ground of the lowest violations of the experimental constraints only, and the phi-psi values for each residue are examined. In the second and final run, the database-derived phi-psi constraints are enforced only for those residues which in the first run have ended in one and the same favored phi-psi region. For residues which are either spread over different favored regions or concentrated in disallowed regions, the constraints are not enforced. The final family is then selected, after the second run, again only based on the agreement with the experimental constraints. This automated approach was implemented in DYANA and was tested on as many as 12 proteins, including some containing paramagnetic metals, whose structures had been previously solved in our laboratory. The quality of the structures, and of Ramachandran plot statistics in particular, was notably improved while preserving the agreement with the experimental constraints.

Computer Simulation↗
PubMed2002

Paramagnetically induced residual dipolar couplings for solution structure determination of lanthanide binding proteins.

Lanthanides may substitute calcium in calcium-binding proteins, such as, for instance, EF-hand proteins. Paramagnetic lanthanides are capable of orienting the protein in high magnetic fields to an extent similar to that obtained by using orienting devices, and each lanthanide orients according to its magnetic susceptibility tensor. Here, Ce(3+), Tb(3+), Dy(3+), Ho(3+), Er(3+), Tm(3+), Yb(3+) in the C-terminal site of calbindin D(9k) have been investigated. Such systems provide (1)H-(15)N residual dipolar couplings (rdc) which can be used for solution structure determinations. Within the frame of optimizing the use of residual dipolar couplings for efficient solution structure determination, it is proposed here to use a number of lanthanides (e.g., >2) to obtain the orientations of the internuclear vectors with respect to an arbitrary reference system. This is facilitated by the independent knowledge of the magnetic susceptibility anisotropy tensor of each metal, obtained from the analysis of the pseudocontact shifts. A further module of the program PARAMAGNETIC-DYANA, called RDCDYANA-ANGLES, is developed to efficiently incorporate such rdc-derived orientations, instead of the rdc themselves, as constraints in the solution structure calculation. This strategy is absolutely general and can be extended to any other pair of dipole-dipole coupled nuclei. The effect of mobility is also assessed. In principle, information on the mobility can be obtained with a number of lanthanide ions >5, or by combining a smaller number of lanthanide ions with a few orienting devices.

Anisotropy↗
PubMed2002

Molecular dynamics characterization of the C2 domain of protein kinase Cbeta.

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. BetaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca(2+) ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca(2+) ions, to two Ca(2+) ions, and in the Ca(2+)-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca(2+) ions. The results show that calcium stabilizes the beta-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca(2+) ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca(2+) ions are able to interact with two molecules of O-phospho-l-serine. These data suggest that Ca(2+) ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation.

Amino Acid Sequence↗
PubMed2002

Cross correlation rates between Curie spin and dipole-dipole relaxation in paramagnetic proteins: the case of cerium substituted calbindin D9k.

Cross correlation rates between Curie spin relaxation and H-N dipole-dipole coupling (gamma(HM,HN)CS,DD) have been determined for a calcium binding protein, Calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). Gamma(HM,HN)CS,DD values depend on both the metal-to-proton distances and the M-H-N angles and can be used as an additional constraint in order to refine the solution structure of paramagnetic metalloproteins. For this purpose, we have implemented a new module (CCR-DYANA) in a version of the program DYANA (PARAMAGNETIC-DYANA), which can be used together with other paramagnetism-based constraints such as pseudocontact shifts, residual dipolar couplings and hyperfine based Karplus relationships. This integrated structure calculation protocol has the advantage that different paramagnetic-based constraints are treated by the same algorithm in a way that the efficiency of each class of constraints can be analyzed and compared.

Algorithms↗
PubMed2001

Chemical shift-based constraints for solution structure determination of paramagnetic low-spin heme proteins with bis-His and His-CN axial ligands: the cases of oxidized cytochrome b(5) and Met80Ala cyano-cytochrome c.

The chemical shifts of the methyl protons of protoporphyrin IX, which are readily assigned, are related to the structural features of the axial histidine ligands in heme proteins with bis-His or His-CN axial coordination (Bertini I, Luchinat C, Parigi G, Walker FA (1999) JBIC 4:515-519). In the present paper, a module is developed which transforms the chemical shifts into a pseudo-potential energy that is a function of the dihedral angles defining the orientation of the axial ligand planes. Minimization of this pseudo-potential energy, together with the energetic contributions provided by the other constraints, yields structures consistent with the heme methyl chemical shifts. Oxidized cytochrome b(5) from rat and the cyanide derivative of the M80A mutant of yeast cytochrome c are used for test calculations. In the case of scarcity of NOEs for the axial ligands, owing to the presence of the paramagnetic center, the above structural constraints are shown to be quite precious. The newly refined structures are deposited in the PDB.

Alanine↗