Structure of nucleophosmin DNA-binding domain and analysis of its complex with a G-quadruplex sequence from the c-MYC promoter.

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a key role in several cellular functions, including ribosome maturation and export, centrosome duplication, and response to stress stimuli. More than 50 mutations at the terminal exon of the NPM1 gene have been identified so far in acute myeloid leukemia; the mutated proteins are aberrantly and stably localized in the cytoplasm due to high destabilization of the NPM1 C-terminal domain and the appearance of a new nuclear export signal. We have shown previously that the 70-residue NPM1 C-terminal domain (NPM1-C70) is able to bind with high affinity a specific region at the c-MYC gene promoter characterized by parallel G-quadruplex structure. Here we present the solution structure of the NPM1-C70 domain and NMR analysis of its interaction with a c-MYC-derived G-quadruplex. These data were used to calculate an experimentally restrained molecular docking model for the complex. The NPM1-C70 terminal three-helix bundle binds the G-quadruplex DNA at the interface between helices H1 and H2 through electrostatic interactions with the G-quadruplex phosphate backbone. Furthermore, we show that the 17-residue lysine-rich sequence at the N terminus of the three-helix bundle is disordered and, although necessary, does not participate directly in the contact surface in the complex.

Sco proteins are involved in electron transfer processes.

Sco proteins are widespread in eukaryotic and in many prokaryotic organisms. They have a thioredoxin-like fold and bind a single copper(I) or copper(II) ion through a CXXXC motif and a conserved His ligand, with both tight and weak affinities. They have been implicated in the assembly of the Cu(A) site of cytochrome c oxidase as copper chaperones and/or thioredoxins. In this work we have structurally characterized a Sco domain which is naturally fused with a typical electron transfer molecule, i.e., cytochrome c, in Pseudomonas putida. The thioredoxin-like Sco domain does not bind copper(II), binds copper(I) with weak affinity without involving the conserved His, and has redox properties consisting of a thioredoxin activity and of the ability of reducing copper(II) to copper(I), and iron(III) to iron(II) of the cytochrome c domain. These findings indicate that the His ligand coordination is the discriminating factor for introducing a metallochaperone function in a thioredoxin-like fold, typically responsible for electron transfer processes. A comparative structural analysis of the Sco domain from P. putida versus eukaryotic Sco proteins revealed structural determinants affecting the formation of a tight-affinity versus a weak-affinity copper binding site in Sco proteins.

Toward structural dynamics: protein motions viewed by chemical shift modulations and direct detection of C'N multiple-quantum relaxation.

Multiple quantum relaxation in proteins reveals unexpected relationships between correlated or anti-correlated conformational backbone dynamics in alpha-helices or beta-sheets. The contributions of conformational exchange to the relaxation rates of C'N coherences (i.e., double- and zero-quantum coherences involving backbone carbonyl (13)C' and neighboring amide (15)N nuclei) depend on the kinetics of slow exchange processes, as well as on the populations of the conformations and chemical shift differences of (13)C' and (15)N nuclei. The relaxation rates of C'N coherences, which reflect concerted fluctuations due to slow chemical shift modulations (CSMs), were determined by direct (13)C detection in diamagnetic and paramagnetic proteins. In well-folded proteins such as lanthanide-substituted calbindin (CaLnCb), copper,zinc superoxide dismutase (Cu,Zn SOD), and matrix metalloproteinase (MMP12), slow conformational exchange occurs along the entire backbone. Our observations demonstrate that relaxation rates of C'N coherences arising from slow backbone dynamics have positive signs (characteristic of correlated fluctuations) in beta-sheets and negative signs (characteristic of anti-correlated fluctuations) in alpha-helices. This extends the prospects of structure-dynamics relationships to slow time scales that are relevant for protein function and enzymatic activity.

The solution structure of the monomeric copper, zinc superoxide dismutase from Salmonella enterica: structural insights to understand the evolution toward the dimeric structure.

The structure of the SodCII-encoded monomeric Cu, Zn superoxide dismutase from Salmonella enterica has been solved by NMR spectroscopy. This represents the first solution structure of a natural and fully active monomeric superoxide dismutase in solution, providing information useful for the interpretation of the evolutional development of these enzymes. The protein scaffold consists of the characteristic beta-barrel common to the whole enzyme family. The general shape of the protein is quite similar to that of Escherichia coli Cu, Zn superoxide dismutase, although some differences are observed mainly in the active site. SodCII presents a more rigid conformation with respect to the engineered monomeric mutants of the human Cu, Zn superoxide dismutase, even though significant disorder is still present in the loops shaping the active site. The analysis of both dynamics and hydration properties of the protein in solution highlights the factors required to maintain the fully active and, at the same time, monomeric protein. This study provides novel insights into the functional differences between monomeric and dimeric bacterial Cu, Zn superoxide dismutases, in turn helping to explain the convergent evolution toward a dimeric structure in prokaryotic and eukaryotic enzymes of this class.

NMR assignment of reduced form of copper, zinc superoxide dismutase from Salmonella enterica.

Almost complete assignment (97%) of NMR resonances was obtained for the reduced, Cu(I), form of prokaryotic CuZnSOD from Salmonella enterica. 13C direct detection was used to complement the standard bouquet of 1H detected triple resonance experiments and contributed to the identification of proline backbone resonances and to side chains assignments of Asx, Glx and aromatic rings. This is the only complete assignment available for monomer SOD from prokaryotic organisms.