Solution structure of the apo and copper(I)-loaded human metallochaperone HAH1

The human metallochaperone HAH1 has been produced in Escherichia coli with four additional amino acids at the C-terminus and characterized in solution by NMR spectroscopy, both with and without copper(I). The solution structure of the apo-HAH1 monomer has a root-mean-square-deviation (RMSD) of 0.50 A for the coordinates of the backbone atoms and 0.96 A for all heavy atoms. These values compare, respectively, with 0.45 and 0.95 A for copper(I)-HAH1. There are only minor structural rearrangements upon copper(I) binding. In particular, the variation of interatomic interactions around the metal-binding region is limited to a movement of Lys60 toward the metal site. The protein structures are similar to those obtained by X-ray crystallography in a variety of derivatives, with backbone RMSD values below 1 A. In the holoprotein, copper(I) is confirmed to be two coordinated. If these data are compared with those of orthologue proteins, we learn that HAH1 has a lower tendency to change coordination number from two to three. Such a switch in coordination is a key step in copper transfer.     

The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status

The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis.     

Bioinformatic comparison of structures and homology-models of matrix metalloproteinases

The entire family of human matrix metalloproteinases (MMPs) was investigated using phylogenetic trees and homology modeling. The phylogenetic analysis indicates that individual domains of each MMP have evolved in a correlated manner. Despite their high sequence similarity, the phylogenetic tree of the catalytic domains already allows functional (e.g., linked to regulation and substrate recognition) homologies between different MMPs to be identified. The same pattern of functional homologies is confirmed by the phylogenetic analysis of the mature proteins. Structural models were built for the catalytic domains of the entire MMP family, for twelve hemopexin domains and for twelve mature proteins. The surface properties around the active site cleft of the modeled and experimental structures are quite conserved, whereas the hemopexin domains are more differentiated, possibly indicating a role in determining substrate specificity. The analysis of mature MMPs showed that the area of the interface between the catalytic and hemopexin domains is essentially conserved, with both hydrophobic and hydrophilic amino acids at the interface. The absence of specific conserved interdomain contacts suggests that the interface is tolerant to amino acid replacements, and that there may be a certain degree of plasticity with respect to the reciprocal orientation of the two domains.     

Biodiversity of brachyuran crabs (Crustacea: Decapoda) from non-consolidated sublittoral bottom on the northern coast of São Paulo State, Brazil

The brachyuran community of the coast of São Paulo State is represented by about 188 species of crabs inhabiting different kinds of coastal marine environments. The biodiversity of brachyurans found on non-consolidated sublittoral bottom was investigated. The Ubatuba region (Ubatumirim, Ubatuba and Mar Virado bays, Couves and Mar Virado Islands, offshore region) was sampled for 3 years (1998–2000), at depths of 2–40 m. All sampling was performed using a fishing boat equipped with two double-rig nets. We collected 79 brachyuran species representing 9 superfamilies (4 Dromioidea, 1 Homoloidea, 2 Calappoidea, 5 Leucosioidea, 20 Majoidea, 7 Parthenopoidea, 17 Portunoidea, 18 Xanthoidea, and 5 Pinnotheroidea) and 41 genera. Ubatuba bay showed the greatest species richness with 50 species, followed by Ubatumirim with 45 and Mar Virado with 29. The number of species collected represents about 57% of the known species of crabs already reported for the shore of São Paulo State. It is worth noticing that this percentage is restricted only to non-consolidated sublittoral bottom. This fact indicates a great biodiversity of the habitat in this studied region, probably one to the diversity of habitat types present in the bays.     

Structural consequences of b- to c-type heme conversion in oxidized Escherichia coli cytochrome b562

An NMR characterization of the 98Arg --> Cys variant of iron (III)-containing cytochrome b562 from Escherichia coli has been performed and the solution structure obtained. This variant has a covalent bond between the heme and Cys 98, thus mimicking the heme binding in cytochrome c. The R98C cytochrome is shown to have a significantly increased stability, compared to that of wild type, toward thermal and chemical denaturation. In water at 20 degrees C it is 5.60 kJ mol-1 more stable than the WT protein, measured by equilibrium guanidine hydrochloride denaturation. The structure has been obtained through two-dimensional total correlation spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments and through three-dimensional NOESY-15N heteronuclear multiple quantum coherence (HMQC). By these methods, 85% of protons and 100% of backbone nitrogens were assigned. 2145 meaningful nuclear Overhauser effects (NOEs) (20 NOEs per residue), 45 backbone 3J values, and 397 pseudocontact shifts were used to obtain a family of 35 members, which were then energy-minimized. The root-mean-square deviation (RMSD) with respect to the average structure is 0.50 +/- 0.07 for the backbone and 1.01 +/- 0.08 for the heavy atoms. The magnetic anisotropy resulting from analysis of the pseudocontact shifts indicates an anisotropy that is an intermediate between that of the wild-type, which is the smallest, and cytochrome c. The g values confirm a higher anisotropy of the variant with respect to the wild-type protein. The chirality of the heme 2 alpha carbon is the same as that in all naturally occurring cytochromes c. The overall secondary structure and tertiary structure are very similar to the wild type. The removal of Arg 98 causes a change in the pH-dependent properties. The pKa, proposed to be due to deprotonation of the coordinated histidine, is 1.5 units higher than in the wild type, consistent with the lack of the positive charge of Arg 98 close to the ionizable group. This is further support for the coordinated histidine being the titratable group with an alkaline pKa in the wild-type protein. The pattern of the shifts of the heme methyl groups is different than in the wild-type protein, presumably due to alteration of the electronic structure by the presence of the covalent bond between the protein and the heme. The difference in stability between the variant and wild-type protein is discussed in terms of the structural information.     

Neutral mixed-ligand complexes of 2,2′-bipyridinedichloroplatinum(II) with dianionic aromatic chelating ligands as potential photosensitizers

The electronic spectra of NCS^? and I^? adducts of cobalt(II) human carbonic anhydrase I are pH dependent at pH values below 7. The pK_a of such equilibrium is dependent on the anion concentration and varies between 4.6 and 6.6. The ¹H NMR spectra show that the three histidine residues are bound to the metal ion over the entire pH range investigated. It is supposed that a Glu residue triggers the change in stereochemistry around the metal ion. It is possible that such a Glu residue is Glu 106 present in the active cavity.     

A new submicroscopic deletion that refines the 9p region for sex reversal

Male to female sex reversal has been described in patients with deletions of chromosome 9p, and a region critical for sex reversal has been localized to p24.3, at the tip of the chromosome (TD9). It was proposed that the sex reversal may arise by haploinsufficiency for a gene localized to the minimum deletion. The 9p24.3 genes DMRT1 and DMRT2 are the favorite TD9 candidates to date, in virtue of their sequence similarity to doublesex and mab-3, sexual regulators in Drosophila and Caenorhabditis elegans, respectively. The hypothesis of sex reversal by combined haploinsufficiency for the two genes was put forward to explain the lack of mutations in either gene in XY sex-reversed females. Here we describe a XY sex-reversed patient carrying a novel 9p deletion that extends over less than 700 kb of genomic DNA. This region defines the smallest interval for sex reversal found to date. DMRT1 and DMRT2 map outside this region. Our data do not support the hypothesis of combined haploinsufficiency for DMRT1 and DMRT2. Nevertheless, DMRT1 localizes very close to the deletion breakpoint and has a pattern of expression compatible with a role in sex determination. It therefore remains a candidate gene for 9p sex reversal.     

Speeding up sequence specific assignment of IDPs

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.     

An idic(15) associated with POF (premature ovarian failure): molecular cytogenetic definition of a case and review of the literature

We report on a 36-year-old infertile woman, presenting a premature ovarian failure with an otherwise normal female phenotype. Cytogenetic analyses showed the presence of a supernumerary marker chromosome, that was characterized by FISH (fluorescent in situ hybridization) and array CGH (comparative genomic hybridization). This marker chromosome was derived from chromosome 15, and contained only heterochromatic material. The Prader Willi/Angelman region was not present. No duplications of the 15q regions were detected by array CGH. Supernumerary markers of chromosome 15 have been reported in cases of infertility and amenorrhea, that is also described in cases with marker derived by other acrocentric chromosomes. The case here presented constitutes a further example that etiology of POF is not always associated with a defective gene, but in some cases oocytes atresia can be the consequence of the abnormal meiotic pairing of chromosomes.