Elena Molteni

Molecular dynamics study of the Cu2+ binding-induced "structuring" of the N-terminal domain of human prion protein.

In this work, we report molecular dynamics simulations on a fragment of the human prion protein spanning residues 31-120, with copper(II) bound to the repeat region in several ways corresponding to the known intra- and inter-repeat coordination modes, or to the metal site located at His111. The results of this study point to a different structuring tendency of the protein fragment depending on copper binding mode, with the highest degree of structuring in the case of intrarepeat Cu(II) coordination corresponding to high copper concentration.

Effect of CuII on the Complex between Kanamycin A and the Bacterial Ribosomal A Site

The solution structure of kanamycin A interacting with a ribosomal A‐site fragment was solved by transferred‐NOE techniques and found to agree with the structure of the complex observed in the crystal. Despite the fast exchange conditions found for the interaction, the bound form was identified by NOESY spectroscopy. At 600 MHz, NOE effects are only observed for the RNA‐associated antibiotic. Dissociation constants were measured by NMR spectroscopy for two sites of interaction (Kd1=150±40 μM; Kd2=360±50 μM). Furthermore, the effects of the CuII ion on the antibiotic, on the RNA fragment that mimics the bacterial ribosomal A site, and on the complex formed between these two entities were analyzed. The study led to the proposal of a model that localizes the copper ion within the kanamycin–RNA complex.

Fine tuning the structure of the Cu2+ complex with the prion protein chicken repeat by proline isomerization

The interaction between the single hexarepeat unit of chicken prion protein [ChPrP(54-59)] and Cu(II) was investigated by NMR, finding different coordination modes for the trans/trans and cis/trans isomers.

The structure of the Ce(III)–Angiotensin II complex as obtained from NMR data and molecular dynamics calculations

Angiotensin II is shown by nuclear magnetic resonance (NMR) to form a complex in water at pH 4.0 with cerium(III), the ideal paramagnetic probe for Ca(2+). Paramagnetic shifts induced by the metal were used for the determination of dissociation constant and complex stoichiometry. ROESY cross-peaks and 3 J(HN-H)(alpha) coupling constants were converted into distance and angular constraints to determine the structure of the complex by molecular dynamics using the simulated annealing protocol. The complex is kinetically labile and involves the Asp-1 side chain and the Phe-8 terminal carboxylates as binding groups resembling a hairpin which has been suggested as a possible biologically active structure.

Structural features of the Cu2+ complex with the rat Aβ(1-28) fragment

The interaction between Cu(II) and the rat amyloid beta (1-28) fragment in micellar solutions at pH 7.5 was investigated by CD and NMR spectroscopy; the proton-copper distances were used in restrained molecular dynamics simulations to obtain a structural model of the Cu(II) complex.

Structural features and oxydative stress towards plasmid DNA of apramycin copper complex

The interaction of apramycin with copper at different pH values was investigated by potentiometric titrations and EPR, UV-vis and CD spectroscopic techniques. The Cu(II)-apramycin complex prevailing at pH 6.5 was further characterized by NMR spectroscopy. Metal-proton distances derived from paramagnetic relaxation enhancements were used as restraints in a conformational search procedure in order to define the structure of the complex. Longitudinal relaxation rates were measured with the IR-COSY pulse sequence, thus solving the problems due to signal overlap. At pH 6.5 apramycin binds copper(II) with a 2 : 1 stoichiometry, through the vicinal hydroxyl and deprotonated amino groups of ring III. Plasmid DNA electrophoresis showed that the Cu(II)-apramycin complex is more active than free Cu(II) in generating strand breakages. Interestingly, this complex in the presence of ascorbic acid damages DNA with a higher yield than in the presence of H(2)O(2).

Structural Features of Apramycin Bound at the Bacterial Ribosome A Site as Detected by NMR and CD Spectroscopy

The ribosome is generally agreed to represent the main target for aminoglycoside and other antibiotics that interact with the decoding site (A site) in 16S rRNA and, therefore, typically decrease the fidelity of translation. NMR spectroscopy and Xray crystallography have revealed the molecular basis for bacterial target discrimination of the natural products, which share a common 2-deoxystreptamine scaffold involved in RNA recognition. Apramycin (Figure 1 A), which is particularly active against Gram-negative bacteria, 9] possesses unique features, such as its peculiar structure containing the unusual pyrano-pyranose sugar and the 2-deoxystreptamine (2-DOS) monosubstituted at 4-position. The unusual mode of action elicits : 1) blockage of ribosome translocation from the aminoacyl acceptor site (A site) to the peptidyl donor site (P site), and 2) the binding of both the bacterial and eukaryotic decoding site rRNA with similar affinities. Moreover, recent crystallographic experiments have shown that apramycin can bind to the A site of the Homo sapiens cytoplasmic ribosome, and that it stabilizes a pre-existing conformational state of the free eukaryotic decoding site rRNA, 17] in contrast to the case of bacterial RNA, in which aminoglycoside binding usually causes dramatic conformational changes. 18] By investigating the solution structure of kanamycin A interacting with a ribosomal A-site fragment, transferred-NOE techniques were found to provide a very convenient method. In fact, although the fast exchange regime in the NMR time scale yields outputs averaged over the free and bound configurations, the absence of NOE connectivities of the free form at 600 MHz allows direct and immediate observation of features related to the RNA-associated antibiotic. Here we present the extension of the same approach to delineation of structural features of apramycin bound to the A site. The assigned H NMR spectrum of apramycin at pH 6.5 (pD 6.9) is shown in Figure 1 A, and is in substantial agreement with data in the literature. 21] Upon addition of 0.05 equiv of RNA, apramycin binding yielded: 1) substantial line broadening, 2) changes in chemical shift (Figure 1 B), in which ring I is apparently the most affected, and 3) appearance of new NOESY spectra (Figure 2). Starting from the tumbling regime experienced in the free state that yields negligible NOE effects at 600 MHz, the relatively slow motion of the complex determines strong cross-peaks originating from the bound form. Analysis of the NOESY spectrum in the presence of RNA (Figure 2 B) is thus a direct probe of the bound conformation of the antibiotic. This could be acquired by obtaining geometrical restraints and by using a conformational search routine in Hyperchem. The intensities of ROESY cross-peaks, referenced to cross-peaks related to proton pairs at fixed distances, were converted into interproton distance restraints. For the free apramycin (Figure S1 A in the Supporting Information) also dihedral angle restraints were used, obtained from J coupling constants (Table S1 in the Supporting Information) through the Haasnoot–Altona equation. 27] Since this equation yields multiple values, we considered only those that were compatible with the values assumed in the possible ring conformations. The H-bond in ring III of free apramycin revealed by NMR spectroscopy data was imposed through a distance restraint of 0.18 nm between the hydroxyl proton and the amino nitrogen. The bound structure is shown in Figure S1 B (in the Supporting Information) with the crystallographic structure superimposed. The bound form maintains the relative orientation of the rings; this indicates, as expected, that the interaction does not require significant molecular rearrangements. Aminoglycosides have been in fact shown to rarely change their conformation upon interaction with their targets, since this process represents a penalty for the affinity to the target. The changes in chemical shifts for apramycin (Dd, ppm) upon interaction with the RNA fragment (Figure 1 B) correlate well with the pattern of interaction found in the crystallographic study. The negative sign of Dd observed on ring II can in fact be explained by its stacking over the guanine base 1491. The CD spectrum of the RNA fragment shows the main features of the double-stranded A-form RNA (Figure 3 A). Upon adding apramycin, the effect experienced by the band at 209 nm was considered for estimation of Kd [30] by plotting L (=Deobs Def, observed CD of bound and free RNA, respectively) versus the free apramycin concentration (Figure 3 B). Standard regression analysis provided Kd = (23.05 3.84) mm for a 1:2 complex stoichiometry, which probably reflects the occurrence of “specific” and “unspecific” interactions. The measured Kd should be therefore taken as an average between the two interaction modes, but it is about one order of magnitude larger than that evaluated by mass spectrometry. [a] Dr. D. Balenci, Dr. N. D’Amelio, Prof. E. Gaggelli, Dr. N. Gaggelli, Dr. E. Molteni, Prof. G. Valensin Department of Chemistry, University of Siena Via A. Moro, 53100 Siena (Italy) Fax: (+ 39) 0577-234233 E-mail : valensin@unisi.it [b] Dr. L. Cellai Istituto di Cristallografia, CNR Area di Ricerca di Roma 1, 00016 Monterotondo Stazione (Italy) [c] Dr. N. D’Amelio Imaging S.p.A. , CRB-Area Science Park Basovizza, Trieste (Italy) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900345

Probing the role of metal ions on reversible peptide–protein interactions by NMR

This work provides evidence that paramagnetic lanthanide ions constitute ideal probes suitable for investigations of metal effects upon peptide–receptor interactions with the use of NMR methods. Cerium(III) is herein used for assessing metal effects upon the interaction between angiotensin II and a fragment from the AT1A receptor. Angiotensin II forms a complex with cerium(III) in water while the fCT300–320 receptor fragment is poorly affected by cerium(III). However, the addition of the fragment displaces cerium(III) from the complex, thus directly demonstrating the higher affinity of angiotensin II for the receptor and probing the peptide residues involved in receptor binding.

NMR structural model of the interaction of herbicides with the photosynthetic reaction center from Rhodobacter sphaeroides.

The interaction of the herbicides acifluorfen and paraquat with the photosynthetic reaction center from Rhodobacter sphaeroides has been studied by NMR relaxation measurements. Interaction in aqueous solution has been demonstrated by evaluating motional features of the bound form through cross‐relaxation terms of protons at fixed distances on the herbicides. Contributions to longitudinal nonselective relaxation rates different from the proton–proton dipolar relaxation were inferred, most probably due to paramagnetic effects originating from the high‐spin nonheme FeII ion in the reaction center. Paramagnetic contributions to proton relaxation rates were converted into distance constraints in order to build a model for the interaction. The models place paraquat in the QB site, where most herbicides interact, in agreement with docking calculations, whereas acifluorfen was placed between the metal and the QB site, as also demonstrated by the induced paramagnetic shifts. Acifluorfen could therefore act to break the electron‐transfer pathway between the QA and QB sites.