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SARS-CoV-2 Mproinhibition by a zinc ion: structural features and hints for drug design

The first structure of the SARS-CoV-2 main protease in complex with an isolated zinc ion provides solid ground for the design of potent and selective metal-conjugated inhibitors

Solution Structure of the Yeast Copper Transporter Domain Ccc2a in the Apo and Cu(I)-loaded States

Ccc2 is an intracellular copper transporter in Saccharomyces cerevisiae and is a physiological target of the copper chaperone Atx1. Here we describe the solution structure of the first N-terminal MTCXXC metal-binding domain, Ccc2a, both in the presence and absence of Cu(I). For Cu(I)-Ccc2a, 1944 meaningful nuclear Overhauser effects were used to obtain a family of 35 structures with root mean square deviation to the average structure of 0.36 +/- 0.06 A for the backbone and 0.79 +/- 0.05 A for the heavy atoms. For apo-Ccc2a, 1970 meaningful nuclear Overhauser effects have been used with 35 (3)J(HNHalpha) to obtain a family of 35 structures with root mean square deviation to the average structure of 0.38 +/- 0.06 A for the backbone and 0.82 +/- 0.07 A for the heavy atoms. The protein exhibits a betaalphabetabetaalphabeta, ferrodoxin-like fold similar to that of its target Atx1 and that of a human counterpart, the fourth metal-binding domain of the Menkes protein. The overall fold remains unchanged upon copper loading, but the copper-binding site itself becomes less disordered. The helical context of the copper-binding site, and the copper-induced conformational changes in Ccc2a differ from those in Atx1. Ccc2a presents a conserved acidic surface which complements the basic surface of Atx1 and a hydrophobic surface. These results open new mechanistic aspects of copper transporter domains with physiological copper donor and acceptor proteins

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

Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy

: In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments. 19F NMR can overcome this issue, as fluorine atoms incorporated in proteins can be detected by simple background-free 1D NMR spectra. Here, we show that fluorinated amino acids can be easily incorporated in proteins expressed in human cells by employing a medium switch strategy. This straightforward approach allows the incorporation of different fluorinated amino acids in the protein of interest, reaching fluorination efficiencies up to 60%, as confirmed by mass spectrometry and X-ray crystallography. The versatility of the approach is shown by performing 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N in-cell NMR. We apply the approach to observe the interaction between an intracellular target, carbonic anhydrase 2, and its inhibitors, and to investigate how the formation of a complex between superoxide dismutase 1 and its chaperone CCS modulates the interaction of the chaperone subunit with the cellular environment

Characterization of the Binding Interface between the Copper Chaperone Atx1 and the First Cytosolic Domain of Ccc2 ATPase

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions

The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded ߠbarrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.Full Tex

Composição química da silagem de diferentes cultivares de Sorgo sacarino (Sorghum bicolor (L.) Moench).

As cultivares de sorgo apresentam, de modo geral, bom valor nutritivo e ampla versatilidade deuso na alimentação animal. Suas características são propicias para a utilização na forma de silagem,entretanto, poucos estudos são realizados para comprovar sua viabilidade. Dessa forma, objetivou-seavaliar a composição bromatológica das silagens de diferentes cultivares de sorgo sacarino. As silagensforam confeccionadas utilizando-se 20 mini-silos de PVC, providos de válvulas do tipo ?Bunsen?. Foramavaliados dois cultivares de sorgo com dois períodos de cultivo (90 e 100 dias), originando quatrotratamentos, caracterizados como: T1 ? Variedade Jovem (90) CMSX 647, T2 ? Variedade Tardia (100)CMSX647,T3?VariedadeJovem(90)BRS506eT4?VariedadeTardia(100)BRS506.Oexperimento foi conduzidosegundo o delineamentointeiramentecasualizado, com 5 repetiçõesportratamento, considerando 5% de probabilidade para o erro tipo I. Os teores de MS foram maiores paraCMSX 100 e BRS 100. De forma inversa, maiores valores de PB foram encontrados para CMSX 90 eBRS 90. As diferentes idades de plantio influenciaram os teores de FDN da silagem. Para EE, FDA, CNFnão houve significância entre os tratamentos. Os cultivares de sorgo sacarino avaliados apresentaram, demodo geral, uma boa composição bromatológica, com potencial para utilização na alimentação animal

Composição química da silagem de diferentes cultivares de Sorgo sacarino (Sorghum bicolor (L.) Moench).

As cultivares de sorgo apresentam, de modo geral, bom valor nutritivo e ampla versatilidade deuso na alimentação animal. Suas características são propicias para a utilização na forma de silagem,entretanto, poucos estudos são realizados para comprovar sua viabilidade. Dessa forma, objetivou-seavaliar a composição bromatológica das silagens de diferentes cultivares de sorgo sacarino. As silagensforam confeccionadas utilizando-se 20 mini-silos de PVC, providos de válvulas do tipo ?Bunsen?. Foramavaliados dois cultivares de sorgo com dois períodos de cultivo (90 e 100 dias), originando quatrotratamentos, caracterizados como: T1 ? Variedade Jovem (90) CMSX 647, T2 ? Variedade Tardia (100)CMSX647,T3?VariedadeJovem(90)BRS506eT4?VariedadeTardia(100)BRS506.Oexperimento foi conduzidosegundo o delineamentointeiramentecasualizado, com 5 repetiçõesportratamento, considerando 5% de probabilidade para o erro tipo I. Os teores de MS foram maiores paraCMSX 100 e BRS 100. De forma inversa, maiores valores de PB foram encontrados para CMSX 90 eBRS 90. As diferentes idades de plantio influenciaram os teores de FDN da silagem. Para EE, FDA, CNFnão houve significância entre os tratamentos. Os cultivares de sorgo sacarino avaliados apresentaram, demodo geral, uma boa composição bromatológica, com potencial para utilização na alimentação animal