Nicola D'Amelio

Bioinorganic Chemistry of Parkinson's Disease: Structural Determinants for the Copper-Mediated Amyloid Formation of Alpha-Synuclein

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinsons disease (PD). A central, unresolved question in the pathophysiology of PD relates to the role of AS-metal interactions in amyloid fibril formation and neurodegeneration. Our previous works established a hierarchy in alpha-synuclein-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. Two independent, non-interacting copper-binding sites were identified at the N-terminal region of AS, with significant difference in their affinities for the metal ion. In this work we have solved unknown details related to the structural binding specificity and aggregation enhancement mediated by Cu(II). The high-resolution structural characterization of the highest affinity N-terminus AS-Cu(II) complex is reported here. Through the measurement of AS aggregation kinetics we proved conclusively that the copper-enhanced AS amyloid formation is a direct consequence of the formation of the AS-Cu(II) complex at the highest affinity binding site. The kinetic behavior was not influenced by the His residue at position 50, arguing against an active role for this residue in the structural and biological events involved in the mechanism of copper-mediated AS aggregation. These new findings are central to elucidate the mechanism through which the metal ion participates in the fibrillization of AS and represent relevant progress in the understanding of the bioinorganic chemistry of PD.

Human SOD1 before Harboring the Catalytic Metal SOLUTION STRUCTURE OF COPPER-DEPLETED, DISULFIDE-REDUCED FORM

SOD1 has to undergo several post-translational modifications before reaching its mature form. The protein requires insertion of zinc and copper atoms, followed by the formation of a conserved S-S bond between Cys-57 and Cys-146 (human numbering), which makes the protein fully active. In this report an NMR structural investigation of the reduced SH-SH form of thermostable E,Zn-as-SOD1 (E is empty; as is C6A, C111S) is reported, characterizing the protein just before the last step leading to the mature form. The structure is compared with that of the oxidized S-S form as well as with that of the yeast SOD1 complexed with its copper chaperone, CCS. Local conformational rearrangements upon disulfide bridge reduction are localized in the region near Cys-57 that is completely exposed to the solvent in the present structure, at variance with the oxidized forms. There is a local disorder around Cys-57 that may serve for protein-protein recognition and may possibly be involved in intermolecular S-S bonds in familial amyotrophic lateral sclerosis-related SOD1 mutants. The structure allows us to further discuss the copper loading mechanism in SOD1.

A paramagnetic probe to localize residues next to carboxylates on protein surfaces

Abstract. It is shown that the paramagnetic properties of lanthanides can be exploited to obtain information on specific parts of a protein surface. Owing to the high affinity of coordinatively unsaturated lanthanide complexes for oxygen donors, carboxylate groups can be used as preferential targets for the interaction. The DO3A ligand is particularly useful in these studies, as it coordinates lanthanides in a heptadentate fashion, leaving two sites available for exogenous donors. A solution of a 15N-labeled sample protein, calbindin D9k (75 residues), was titrated with up to 200% of Gd(III)-DO3A complex, and an inversion recovery 15N-1H HSQC experiment was used to measure the paramagnetic contributions to the longitudinal relaxation rates of the amide protons. Relaxation data were used as distance constraints to estimate the number of interacting complexes and the occupancies of their binding sites. Four preferential interaction sites on the protein surface are found. Inspection of the various carboxylate side chains on the surface of the protein indicates that Gd(III)-DO3A interacts preferentially with carboxylate-rich regions, rather than with isolated carboxylates, suggesting the possibility of chelation of one Gd(III)-DO3A molecule by two carboxylate groups. Gd(III)-DO3A is thus a valuable semi-selective probe for clusters of negative charges on the protein surface.

Metal Ion Effects on the cis/trans Isomerization Equilibrium of Proline in Short-Chain Peptides: A Solution NMR Study

The effect of copper(II) ions on the probabilities of existence of the four detectable conformers of the tetrapeptide Tyr‐Pro‐Phe‐Pro (β‐casomorphin 4) in [2H6]DMSO was investigated by 1H NMR spectroscopy. Integration of the Phe‐NH signals provided the relative populations in the free state as tt/tc/ct/cc=28:34:29:9 at 293 K (c=cis, t=trans). Copper(II) was shown to bind to all four isomers, yielding complexes with two different structures, depending on the conformation of Pro2. The interpretation of paramagnetic relaxation rates of Pro2‐Hα signals provided the corresponding isomeric probabilities in the metal‐bound state as 13:36:20:31. The observed stabilization of the conformation with the lowest probability of existence (cc) may be relevant for the biological role of copper and other metal ions.

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.

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.

Isolation of a 177Hf complex formed by beta-decay of a 177Lu-labeled radiotherapeutic compound and NMR structural elucidation of the ligand and its Lu and Hf complexes.

(177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is being developed for the radiotherapeutic treatment of tumors that express the gastrin-releasing peptide receptor (GRP-R). In this study we investigated the fate of the (177)hafnium ((177)Hf) that forms upon the decay of (177)Lu while the latter is complexed with AMBA. When decayed solutions of (177)Lu-AMBA were analyzed, it was found that (177)Hf is retained in the DO3A monoamide chelator, forming a pair of interconverting isomers. We report the synthesis and full characterization of (nat)Lu-AMBA and the studies performed to demonstrate its correspondence to radioactive (177)Lu-AMBA. We also report the synthesis and characterization of Hf-AMBA and, by NMR studies, show structural analogies between Hf-AMBA, its parent compound Lu-AMBA, and the unmetallated AMBA ligand. In the NMR spectra of both the metallated and unmetallated AMBA ligand, a stacking interaction between the amino benzoyl residue in the linker and a tryptophan in the truncated bombesin [BBN(7-14)-NH(2)] peptide targeting group was found.

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).

Inferences on the nature of a Cr(V) or Cr(IV) species formed by reduction of dichromate by a bovine liver homogenate: NMR and mass-spectrometric studies.

A low-molecular weight chromium-containing fraction of the material resulting from dichromate reduction by bovine liver homogenate was investigated by NMR and ES-MS. The ES-MS spectrum showed a readily detectable peak at m/z = 786.1. The same molecular weight reasonably agreed with the relatively low diffusion coefficient measured by NMR-DOSY experiments on the main species observed in the 1H NMR spectrum. At least two downfield shifted and broad paramagnetic signals were apparent in the 1H NMR spectrum. Temperature dependence of chemical shift was exploited in order to estimate the diamagnetic shift of the signals in the diamagnetic region of the spectrum. 2D TOCSY, NOESY, COSY and 1H-3C HMQC spectra revealed the presence of aromatic protons (which were assigned as His residues), Gly and some other short chain amino-acids. Combinations of the molecular masses of such components together with acetate (which is present in the solution) and chromium atoms allowed a tentative proposal of a model for the compound.

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