CRYSTALLOGRAPHIC STUDIES OF METALLOPROTEINS: CYTOCHROME C2 FROM RHODOPSEUDOMONAS PALUSTRIS AND MAMMALIAN TRANSCOBALAMIN

2001/2002This thesis mainly direct to a few transporter metalloproteins, in particular those containing metallo-organic prosthetic groups (the iron porphyrin and the cobalt corrin complexes ). The three-dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidised state at 1.70 À and in the reduced state at 1.95 À resolution. These are the first highresolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+365 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The positional change of a buried water molecule, located at the heme binding pocket near to the iron bonded Met, is the most prominent structural difference observed with the change of the iron oxidation state in the c-type cytochrome structures. In all oxidised forms this water molecule is found in a position different from that found in the reduced forms. On the contrary, in the oxidised form of the cytochrome c2 from Rhodopseudomonas palustris, this water molecule is detected in a position close to that found in the reduced form. The buried water molecule plays an important role in adjusting the midpoint redox potential of c-type cytochromes with alteration of the surrounding hydrogen bond network. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 310-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an extemal methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles and 'V of about -140 and -130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 À resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron-atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localised change in the backbone conformation, involving mainly the residues Lys92, Met93 and Thr94. Previous studies under typical denaturing conditions, including high pH values and the presence of exogenous ligands, have showed that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia-adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilises the nati ve form, and the presence, in this bacterial cytochrome c2 of the rare six-residue insertion in the helix 310 conformation that increases the Met loop flexibility. Transcobalamin is a cobalamin binding protein in mammalian plasma that facilitates the cellular uptake of vitamin B 12 • Whereas the X-ray structures of severa! B12-enzymes are available, no structural information on B12-transporting proteins has so far been reported. Human and bovine transcobalamin were expressed using ricombinant yeast cells and purified. Human transcobalamin was successfully crystallised usmg polyethylene glycol and ethanol as precipitants. Crystals belong to the orthorhombic space group P212121, with unit-cell parameters a= 49.04, b = 145.27, and e= 164.96 À. A complete data set to 3.2 À resolution was collected from a single crystal using s ynchrotron radiati on (Elettra, Trieste). Estimati on of the crystal packing (V m = 3.2 À 3 Da-1) and self-rotation function analysis suggest the presence of two molecules in the asymmetric unit related by a pseudo-twofold symmetry. Bovine transcobalamin was crystallised using polyethylene glycol and MPD. Crystals belong to the monoclinic space group P21 (a = 95.32, b = 100.19, e = 98.73 À and ~ = 96.9 À), and diffract to better then 2.0 À resolution. MAD data sets were collected at Elettra (Trieste) from a single crystal of the bovine protein, using the cobalamin Co atom as anomalous scatterer. Attempts to find the position of Co atoms in the monoclinic cell from Bijvoet difference Patterson maps are under way.XV Ciclo1973Versione digitalizzata della tesi di dottorato cartacea

Mapping, Structure and Modulation of PPI

Because of the key relevance of protein–protein interactions (PPI) in diseases, the modulation of protein-protein complexes is of relevant clinical significance. The successful design of binding compounds modulating PPI requires a detailed knowledge of the involved protein-protein system at molecular level, and investigation of the structural motifs that drive the association of the proteins at the recognition interface. These elements represent hot spots of the protein binding free energy, define the complex lifetime and possible modulation strategies. Here, we review the advanced technologies used to map the PPI involved in human diseases, to investigate the structure-function features of protein complexes, and to discover effective ligands that modulate the PPI for therapeutic intervention

Bile Acid Recognition by NAPE-PLD

The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide and other lipid signaling amides, including oleoylethanolamide and palmitoylethanolamide. These bioactive molecules play important roles in several physiological pathways including stress and pain response, appetite and lifespan. Recently, we reported the crystal structure of human NAPE-PLD and discovered specific binding sites for the bile acid deoxycholic acid. In this study we demonstrate that in the presence of this secondary bile acid, the stiffness of the protein measured by elastic neutron scattering increases, and NAPE-PLD results ~7 times faster to catalyze the hydrolysis of the more unsaturated substrate N-arachidonyl-phosphatidylethanolamine, compared with N-palmitoyl-phosphatidylethanolamine. Chenodeoxycholic acid and glyco- or tauro-dihydroxy conjugates can also bind to NAPE-PLD and drive its activation. The only natural monohydroxy bile acid, lithocholic acid, shows an affinity of ~20 μM and acts instead as a reversible inhibitor (IC(50) ≈ 68 μM). Overall, these findings provide important insights into the allosteric regulation of the enzyme mediated by bile acid cofactors, and reveal that NAPE-PLD responds primarily to the number and position of their hydroxyl groups

A metallo-beta-lactamase enzyme in action: Crystal structures of the monozinc carbapenemase CphA and its complex with biapenem

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity. (C) 2004 Elsevier Ltd. All rights reserved

Comparison of 99mTc-Labeled Colloid SPECT/CT and Planar Lymphoscintigraphy in Sentinel Lymph Node Detection in Patients with Melanoma: A Meta-Analysis

We compared the detection rate (DR) for sentinel lymph nodes (SLNS), the number of SLNs and the subjects with additional SLNs of single-photon emission computed tomography (SPECT/CT) and planar lymphoscintigraphy (PL) in patients with melanoma. Furthermore, we evaluated the impact of SPECT/CT on surgical plans. Articles containing head-to-head comparisons between SPECT/CT and PL were searched in Pubmed/MEDLINE and Scopus. The literature search was updated until December 31st, 2019. DR was calculated on a per-patient-based analysis; the studies were pooled by their odds ratios (ORs) with a random effects model to assess the significance of difference (p< 0.05). The number of additional SLNs (calculated as the relative risk) and pooled proportion of patients with additional SLNs were investigated. The pooled ratio of surgical procedures influenced by the SPECT/CT findings was calculated. Seventeen studies with 1438 patients were eligible for the calculation of DR of SPECT/CT and PL. The average DR was 98.28% (95% confidence interval (95% CI): 97.94-99.19%) for the SPECT/CT and 95.53% (95% CI: 92.55-97.77%) for the PL; OR of 2.31 (95% CI: 1.66-4.18,p< 0.001) in favor of the SPECT/CT. There was a relative risk of a higher number of SLNs (1.13) for the SPECT/CT and 17.87% of patients with additional SLNs were detected by SPECT/CT. The average impact of SPECT/CT on surgery resulted in 37.43% of cases. This meta-analysis favored SPECT/CT over PL for the identification of SLNs in patients with melanoma due to a higher DR, reproducibility, number of SLNs depicted, proportion of patients with additional SLNs and the impact on the surgical plan. However, PL remains a good option due to the high values of the DR for SLNs