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Biomedical subjects

Teresa Carlomagno

Publications and source records attributed to Teresa Carlomagno.

At least 19 recordsLinked to original sources
PubMed2006

Stereochemical determination of Archazolid A and B, highly potent vacuolar-type ATPase inhibitors from the Myxobacterium Archangium gephyra.

[structure: see text] The relative and absolute stereochemistry of the structurally unique 24-membered myxobacterial macrolides archazolid A and B, highly potent vacuolar-type ATPase (V-ATPase) inhibitors in vitro and in vivo, was determined on the basis of a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and chemical methods.

Antimitotic Agents↗
PubMed2006

SHARP-TACSY: triple-band tailored correlated spectroscopy for base-to-sugar transfer in nucleic acid residues with intermediate time scale motions.

Established experiments to identify the sugar-to-base connectivity in isotopically labeled RNA require long transfer periods and are inefficient for residues undergoing intermediate time scale motions (microsecond to millisecond). Here, an alternative transfer experiment is introduced, whereby the C1'-N1/9-C6/8 spin system is selectively brought to the so-called Hartmann-Hahn condition using selectiveheteronuclear planar triple-band tailored correlated spectroscopy (SHARP-TACSY). Results are shown for the fully labeled 30-mer oligonucleotide TAR RNA with particular attention placed on residues from and close to the bulge and the loop. For these residues, the faster relaxation can be attributed to exchange contributions stemming from transient stacking and unstacking of the bases and/or from the isomerization of the ribose sugar pucker. The new experiment shows improved signal-to-noise for residues exhibiting large microsecond-millisecond time scale motions with respect to established experiments, thus providing a valid alternative for resonance assignment in mobile RNA regions.

Carbohydrates↗
PubMed2006

TAR-RNA recognition by a novel cyclic aminoglycoside analogue.

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.

Aminoglycosides↗
PubMed2006

A thorough dynamic interpretation of residual dipolar couplings in ubiquitin.

The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822-5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098-6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible.

Humans↗
PubMed2005

Influence of the 2'-hydroxyl group conformation on the stability of A-form helices in RNA.

The 2'-hydroxyl group plays fundamental roles in both the structure and the function of RNA, and is the major determinant of the conformational and thermodynamic differences between RNA and DNA. Here, we report a conformational analysis of 2'-OH groups of the HIV-2 TAR RNA by means of NMR scalar coupling measurements in solution. Our analysis supports the existence of a network of water molecules spanning the minor groove of an RNA A-form helix, as has been suggested on the basis of a high-resolution X-ray study of an RNA duplex. The 2'-OH protons of the lower stem nucleotides of the TAR RNA project either towards the O3' or towards the base, where the 2'-OH group can favorably participate in H-bonding interactions with a water molecule situated in the nucleotide base plane. We observe that the k(ex) rate of the 2'-OH proton with the bulk solvent anti-correlates with the base-pair stability, confirming the involvement of the 2'-OH group in a collective network of H-bonds, which requires the presence of canonical helical secondary structure. The methodology and conformational analysis presented here are broadly applicable and facilitate future studies aimed to correlate the conformation of the 2'-OH group with both the structure and the function of RNA and RNA-ligand complexes.

HIV-2↗
PubMed2005

Identification of a novel pharmacophore for peptide toxins interacting with K+ channels.

KappaM-conotoxin RIIIK blocks TSha1 K+ channels from trout with high affinity by interacting with the ion channel pore. As opposed to many other peptides targeting K+ channels, kappaM-RIIIK does not possess a functional dyad. In this study we combine thermodynamic mutant cycle analysis and docking calculations to derive the binding mode of kappaM-conotoxin RIIIK to the TSha1 channel. The final model reveals a novel pharmacophore, where no positively charged side chain occludes the channel pore. Instead the positive-charged residues of the toxin form a basic ring; kappaM-RIIIK is anchored to the K+ channel via electrostatic interactions of this basic ring with the loop and pore helix residues of the channel. The channel amino acid Glu-354 is likely to be a fundamental determinant of the selectivity of kappaM-RIIIK for the TSha1 channel. The Cgamma-OH of Hyp-15 is in contact with the carbonyls of the selectivity filter, disturbing the charge distribution pattern necessary for the coordination of K+ ions. This novel, experimentally based pharmacophore model proves the existence of diverse binding modes of peptidic toxins to K+ channels and underlines the role of intermolecular electrostatic interactions involving channel loop side chains in determining the selectivity of toxins for specific K+ channel types.

Amino Acid Sequence↗
PubMed2005

Assignment and NOE analysis of 2'-hydroxyl protons in RNA: implications for stabilization of RNA A-form duplexes.

The ribose 2'-OH hydroxyl group distinguishes RNA from DNA. The 2'-OH hydroxyl protons are responsible for differences in conformation, hydration, and thermodynamic stability of RNA and DNA oligonucleotides. Additionally, the 2'-OH group plays a central role in RNA catalysis. This important group lies in the shallow groove of RNA, where it is involved in a network of hydrogen bonds with water molecules stabilizing RNA A-form duplexes. Structural and dynamical information on 2'-OH hydroxyl protons is essential to understand their respective roles. Here we report the 2'-OH hydroxyl proton assignments for a 30mer RNA, the HIV-2 transactivation region, in water using solution NMR techniques. We provide structural information on 2'-OH hydroxyl groups in the form of orientational preferences contradicting the paradigm that the 2'-OH hydroxyl typically points away from the ribose H1' proton.

HIV Long Terminal Repeat↗
PubMed2005

Ligand-target interactions: what can we learn from NMR?

The conformation of the ligand in complex with a macromolecular target can be studied by nuclear magnetic resonance (NMR) in solution for both tightly and weakly forming complexes. In the weak binding regime (k(off) > 10(4) Hz), the structure of the bound ligand is accessible also for very large complexes (>100 kDa), which are not amenable to NMR studies in the tight binding regime. Here I review the state-of-the-art NMR methodology used for screening ligands and for the structural investigation of bound ligand conformations, in both tight and weak binding regimes. The advantages and disadvantages of each approach are critically described. The NMR methodology used to investigate transiently forming complexes has expanded considerably in the past few years, opening new possibilities for a detailed description of ligand-target interactions. Novel methods for the determination of the bound ligand conformation, in particular transferred cross-correlated relaxation, are thoroughly reviewed, and their advantages with respect to established methodology are discussed, using the epothilone-tubulin complex as a primary example.

Animals↗
PubMed2004

KappaM-conotoxin RIIIK, structural and functional novelty in a K+ channel antagonist.

Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K(+) channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-Conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K(+) channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K(+) channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of mu-conotoxin GIIIA, a specific blocker of the skeletal muscle Na(+) channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K(+) channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K(+) channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K(+) channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.

Amino Acid Sequence↗
PubMed2004

Structure of the NCoA-1/SRC-1 PAS-B domain bound to the LXXLL motif of the STAT6 transactivation domain.

Signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 (IL-4) by direct interaction with coactivators. The CREB-binding protein (p300/CBP) and the nuclear coactivator 1 (NCoA-1), a member of the p160/steroid receptor coactivator family, bind independently to specific regions of the STAT6 transactivation domain and act as coactivators. The interaction between STAT6 and NCoA-1 is mediated by an LXXLL motif in the transactivation domain of STAT6. To define the mechanism of coactivator recognition, we determined the crystal structure of the NCoA-1 PAS-B domain in complex with the STAT6 LXXLL motif. The amphipathic, alpha-helical STAT6 LXXLL motif binds mostly through specific hydrophobic interactions to NCoA-1. A single amino acid of the NCoA-1 PAS-B domain establishes hydrophilic interactions with the STAT6 peptide. STAT6 interacts only with the PAS-B domain of NCoA-1 but not with the homologous regions of NCoA-2 and NCoA-3. The residues involved in binding the STAT6 peptide are strongly conserved between the different NCoA family members. Therefore surface complementarity between the hydrophobic faces of the STAT6 fragment and of the NCoA-1 PAS-B domain almost exclusively defines the binding specificity between the two proteins.

Amino Acid Motifs↗
PubMed2003

Structural studies on a twin-arginine signal sequence.

Translocation of folded proteins across biological membranes can be mediated by the so-called 'twin-arginine translocation' (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.

Amino Acid Sequence↗
PubMed2003

Measuring the chi 1 torsion angle in protein by CH-CH cross-correlated relaxation: a new resolution-optimised experiment.

Here we introduce an experiment with high sensitivity and resolution for the measurement of CH-CH dipolar-dipolar cross-correlated relaxation rates (CCRR) in protein side-chains. The new methodology aims to the determination of structural and dynamical parameters around the torsion angle chi(1) by measuring C(alpha)H(alpha)-C(beta)H(beta) cross-correlated relaxation rates. The method is validated on the protein ubiquitin: the chi(1) angles determined from the CCRR data are compared with the chi(1) angles of a previously determined NMR structure. The agreement between the two data sets is excellent for most residues. The few discrepancies that were found between the CCR-derived chi(1) angles and the angles of the previously determined NMR structure could be explained by taking internal motion into account. The new methodology represents a very powerful tool to determine both structure and dynamics of protein side-chains in only one experiment.

Magnetic Resonance Spectroscopy↗