Mario Piccioli

Spectroscopic studies on Cu2Zn2SOD: a continuous advancement of investigation tools

Resultats des analyses spectrales effectuees avec diverses techniques spectrometriques dans le but de caracteriser le site actif de complexation de la superoxide dismutase avec les ions Cu 2+ et Zn 2+ . Synthese bibliographique

NMR structures of paramagnetic metalloproteins

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.

A SPECTROSCOPIC CHARACTERIZATION OF A MONOMERIC ANALOG OF COPPER, ZINC SUPEROXIDE DISMUTASE

A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glus, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis.

[2Fe-2S] cluster transfer in iron–sulfur protein biogenesis

Significance Biogenesis of iron–sulfur proteins is a complex process requiring a large number of accessory proteins. In eukaryotes, [2Fe-2S] clusters are synthesized in mitochondria on a scaffold protein. The cluster is then released to monothiol glutaredoxin 5 (GRX5), which was proposed to mediate the transfer of [2Fe-2S] clusters from the scaffold protein to several target proteins, but its precise molecular function remains to be clarified. By investigating the molecular recognition between human GRX5 and its partner proteins (human ISCA1 and ISCA2) and characterizing at the molecular level the cluster transfer process between them, we have shown that a switch between two conformational states of holo GRX5 drives the cluster transfer event, which occurs by a specific protein–protein recognition process. Monothiol glutaredoxins play a crucial role in iron–sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein–protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein–protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.

Locating the metal ion in calcium-binding proteins by using cerium(III) as a probe.

The detection and assignment of NMR spectroscopic signals of carbon atoms from carbonyl and carboxylate groups in the loop hosting the CeIII ion was performed for the cerium‐substituted calcium‐binding protein calbindin D9k. This provided a tool to characterize in solution the first coordination sphere of the metal ion. Due to the well‐documented possibility of replacing calcium with metal ions of the LnIII series, this approach turns out to be extremely efficient for characterizing in solution the coordination of calcium ions in proteins, independently of the availability of X‐ray crystal structures. The present approach completes the structural characterization of lanthanide‐substituted calcium‐binding proteins, for which the role of long‐range constraints arising from hyperfine interaction and self‐orientation has already been assessed.

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA.

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Biogenesis of iron–sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron–sulfur protein assembly machinery, two human key proteins—NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin—form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron–sulfur cluster proteins. The Ndor1–anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.

Mapping the Interaction between the Hemophore HasA and Its Outer Membrane Receptor HasR Using CRINEPT−TROSY NMR Spectroscopy

The first step of heme acquisition by Gram-negative pathogenic bacteria through the so-called heme acquisition system, Has, requires delivery of the heme from the extracellular hemophore protein HasA to a specific outer membrane receptor, HasR. CRINEPT-TROSY NMR experiments in DPC micelles were here used to obtain information on the intermediate HasA-HasR complex in solution. A stable protein-protein adduct is detected both in the presence and in the absence of heme. Structural information on the complexed form of HasA is obtained from chemical shift mapping and statistical analysis of the spectral fingerprint of the protein NMR spectra obtained under different conditions. This approach shows the following: (i) only three different conformations are possible for HasA in solution: one for the isolated apoprotein, one for the isolated holoprotein, and one for the complexed protein, that is independent of the presence of the heme; (ii) the structure of the hemophore in the complex resembles the open conformation of the apoprotein; (iii) the surface contact area between HasA and HasR is independent of the presence of the heme, involving loop L1, loop L2, and the beta2-beta6 strands; (iv) upon complex formation the heme group is transferred from holoHasA to HasR.

Cross correlation rates between Curie spin and dipole-dipole relaxation in paramagnetic proteins: the case of cerium substituted calbindin D9k.

Cross correlation rates between Curie spin relaxation and H-N dipole-dipole coupling (ΓCS,DDHM,HN) have been determined for a calcium binding protein, Calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). ΓCS,DDHM,HNvalues depend on both the metal-to-proton distances and the M-H-N angles and can be used as an additional constraint in order to refine the solution structure of paramagnetic metalloproteins. For this purpose, we have implemented a new module (CCR-DYANA) in a version of the program DYANA (PARAMAGNETIC-DYANA), which can be used together with other paramagnetism-based constraints such as pseudocontact shifts, residual dipolar couplings and hyperfine based Karplus relationships. This integrated structure calculation protocol has the advantage that different paramagnetic-based constraints are treated by the same algorithm in a way that the efficiency of each class of constraints can be analyzed and compared.

ePHOGSY experiments on a paramagnetic protein: location of the catalytic water molecule in the heme crevice of the oxidized form of horse heart cytochrome c

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by 1H NMR spectroscopy. Application of ePHOGSY (enhanced protein hydration observed through gradient spectroscopy) experiments over a paramagnetic molecule provided firm spectroscopic evidence of the presence of a water molecule in the heme crevice. A few intermolecular NOEs have been used to locate the water molecule at about 0.65 nm away from the iron atom and to compare the position observed in solution with that observed in the crystal structure and in solution for the reduced state. The resulting picture is that there is a detectable movement of the water molecule upon oxidation.