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Vicent Esteve

Publications and source records attributed to Vicent Esteve.

7 recordsLinked to original sources
PubMed2007

Structural insights into the GTPase domain of Escherichia coli MnmE protein.

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.

Amino Acid Sequence↗
PubMed2005

Snake venom disintegrins: evolution of structure and function.

Disintegrins represent a family of polypeptides present in the venoms of various vipers that selectively block the function of integrin receptors. Here, we review our current view and hypothesis on the emergence and the structural and functional diversification of disintegrins by accelerated evolution and the selective loss of disulfide bonds of duplicated genes. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information on new structural determinants involved in integrin recognition that may be useful in basic and clinical research. The role of the composition, conformation, and dynamics of the integrin inhibitory loop acting in concert with the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.

Amino Acid Motifs↗
PubMed2005

Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR.

Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.

Animals↗
PubMed2004

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

Amino Acid Sequence↗
PubMed2003

The structural plasticity of the C terminus of p21Cip1 is a determinant for target protein recognition.

The cyclin-dependent kinase inhibitory protein p21(Cip1) might play multiple roles in cell-cycle regulation through interaction of its C-terminal domain with a defined set of cellular proteins such as proliferating cell nuclear antigen (PCNA), calmodulin (CaM), and the oncoprotein SET. p21(Cip1) could be described as an intrinsically unstructured protein in solution although the C-terminal domain adopts a well-defined extended conformation when bound to PCNA. However, the molecular mechanism of the interaction with CaM and the oncoprotein SET is not well understood, partly because of the lack of structural information. In this work, a peptide derived from the C-terminal domain of p21(Cip1) that covers the binding domain of the three above-mentioned proteins was used to demonstrate that the C-terminal domain of p21 recognizes multiple ligands through its ability to adopt multiple conformations. The conformation is dictated by tertiary contacts rather than by the primary sequence of the protein. Our results suggest that the C-terminal domain of p21(Cip1) adopts an extended structure when bound to PCNA and probably when bound to the oncoprotein SET, but an alpha helix when bound to CaM.

Binding, Competitive↗
PubMed2003

NMR study of hexanucleotide d(CCGCGG)2 containing two triplet repeats of fragile X syndrome.

Long repeated stretches of d(CCG) and tri-nucleotide are crucial mutations that cause hereditary forms of mental retardation (fragile X-syndrome). Moreover, the alternating (CG) di-nucleotide is one of the candidates for Z-DNA conformation. Solution NMR structure of d(CCGCGG)(2) has been solved and is discussed. The determined NMR solution structure is a distorted highly bent B-DNA conformation with increased flexibility in both terminal residues. This conformation differs significantly from the Z-DNA tetramer structure reported for the same hexamer in the crystal state at similar ionic strength by Malinina and co-workers. Crystal structure of d(CCGCGG)(2) at high salt concentration includes a central alternating tetramer in Z-DNA conformation, while the initial cytosine swings out and forms a Watson-Crick base-pair with the terminal guanine of a symmetry-related molecule. In solution, NMR data for sugar ring puckering combined with restrained molecular dynamics simulations starting from a Z-DNA form show that terminal furanose residues could adopt the conformation required for aromatic bases swinging out. Therefore, tetramer formation could be considered possible once the hexanucleotide had previously adopted the Z-DNA form. This work gives some insight into correlations between anomalous crystal structures and their accessibility in the solution state.

Computer Simulation↗