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1.
Inorg Chem ; 63(23): 10713-10725, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38805564

RESUMO

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.


Assuntos
Domínio Catalítico , Aprendizado de Máquina , Metaloproteínas , Teoria Quântica , Metaloproteínas/química , Humanos , Modelos Moleculares , Metaloproteinase 12 da Matriz/química , Metaloproteinase 12 da Matriz/metabolismo
2.
J Am Chem Soc ; 145(2): 1389-1399, 2023 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-36604341

RESUMO

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.


Assuntos
Flúor , Chaperonas Moleculares , Humanos , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Flúor/química , Aminoácidos
3.
Angew Chem Int Ed Engl ; 62(31): e202303202, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37276329

RESUMO

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.


Assuntos
Proteínas de Transporte , Imageamento por Ressonância Magnética , Humanos , Preparações Farmacêuticas , Espectroscopia de Ressonância Magnética , Proteínas de Transporte/química , Cristalografia por Raios X
4.
Angew Chem Int Ed Engl ; 60(27): 14960-14966, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33595173

RESUMO

Metalloproteins constitute a significant fraction of the proteome of all organisms and their characterization is critical for both basic sciences and biomedical applications. A large portion of metalloproteins bind paramagnetic metal ions, and paramagnetic NMR spectroscopy has been widely used in their structural characterization. However, the signals of nuclei in the immediate vicinity of the metal center are often broadened beyond detection. In this work, we show that it is possible to determine the coordination environment of the paramagnetic metal in the protein at a resolution inaccessible to other techniques. Taking the structure of a diamagnetic analogue as a starting point, a geometry optimization is carried out by fitting the pseudocontact shifts obtained from first principles quantum chemical calculations to the experimental ones.


Assuntos
Metaloproteínas/química , Fenômenos Magnéticos , Ressonância Magnética Nuclear Biomolecular
5.
J Struct Biol ; 206(1): 99-109, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502494

RESUMO

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metaloproteínas/química , Metais/química , Ressonância Magnética Nuclear Biomolecular/métodos , Compostos Organometálicos/química , Cobre/química , Ferro/química , Modelos Moleculares , Níquel/química , Conformação Proteica
6.
J Biol Inorg Chem ; 24(1): 91-101, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30470900

RESUMO

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.


Assuntos
Anidrase Carbônica II/química , Níquel/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica
7.
Chemistry ; 25(8): 1984-1991, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30462348

RESUMO

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.


Assuntos
Asparaginase , Asparaginase/química , Asparaginase/metabolismo , Materiais Revestidos Biocompatíveis , Espectroscopia de Ressonância Magnética/métodos , Polietilenoglicóis , Multimerização Proteica
8.
Bioorg Med Chem ; 27(24): 115177, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711716

RESUMO

The oxytocinase subfamily of M1 aminopeptidases consists of three members, ERAP1, ERAP2 and IRAP that play several important biological roles, including key functions in the generation of antigenic peptides that drive human immune responses. They represent emerging targets for pharmacological manipulation of the immune system, albeit lack of selective inhibitors is hampering these efforts. Most of the previously explored small-molecule binders target the active site of the enzymes via strong interactions with the catalytic zinc(II) atom and, while achieving increased potency, they suffer in selectivity. Continuing our earlier efforts on weaker zinc(II) binding groups (ZBG), like the 3,4-diaminobenzoic acid derivatives (DABA), we herein synthesized and biochemically evaluated analogues of nine potentially weak ZBGs, based on differential substitutions of functionalized pyridinone- and pyridinethione-scaffolds, nicotinic-, isonicotinic-, aminobenzoic- and hydrazinobenzoic-acids. Crystallographic analysis of two analogues in complex with a metalloprotease (MMP-12) revealed unexpected binding topologies, consistent with the observed affinities. Our results suggest that the potency of the compounds as inhibitors of ERAP1, ERAP2 and IRAP is primarily driven by the occupation of active-site specificity pockets and their proper orientation within the enzymes.


Assuntos
Aminopeptidases/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/antagonistas & inibidores , Zinco/química , Aminopeptidases/metabolismo , Sítios de Ligação , Desenho de Fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Zinco/metabolismo
9.
J Am Chem Soc ; 140(43): 14401-14412, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30269484

RESUMO

The maturation of mitochondrial iron-sulfur proteins requires a complex protein machinery. Human IBA57 protein was proposed to act in a late phase of this machinery, along with GLRX5, ISCA1, and ISCA2. However, a molecular picture on how these proteins cooperate is not defined yet. We show here that IBA57 forms a heterodimeric complex with ISCA2 by bridging a [2Fe-2S] cluster, that [2Fe-2S] cluster binding is absolutely required to promote the complex formation, and that the cysteine of the conserved motif characterizing IBA57 protein family and the three conserved cysteines of the ISCA protein family act as cluster ligands. The [2Fe-2S] heterodimeric complex is the final product when IBA57 is either exposed to [2Fe-2S] ISCA2 or in the presence of [2Fe-2S] GLRX5 and apo ISCA2. We also find that the [2Fe-2S] ISCA2-IBA57 complex is resistant to highly oxidative environments and is capable of reactivating apo aconitase in vitro. Collectively, our data delinate a [2Fe-2S] cluster transfer pathway involving three partner proteins of the mitochondrial ISC machinery, that is, GLRX5, ISCA2 and IBA57, which leads to the formation of a [2Fe-2S] ISCA2-IBA57 complex.

10.
Proc Natl Acad Sci U S A ; 110(18): 7136-41, 2013 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-23596212

RESUMO

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.


Assuntos
Flavoproteínas/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas Ferro-Enxofre/biossíntese , Oxirredutases/biossíntese , Biossíntese de Proteínas , Transporte de Elétrons , Mononucleotídeo de Flavina/metabolismo , Flavoproteínas/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Ferro-Enxofre/química , Modelos Biológicos , Modelos Moleculares , Oxirredutases/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína
11.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 4): 958-67, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24699641

RESUMO

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.


Assuntos
Cristalografia por Raios X/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Domínio Catalítico , Modelos Moleculares , Reprodutibilidade dos Testes
12.
Protein Expr Purif ; 104: 26-33, 2014 12.
Artigo em Inglês | MEDLINE | ID: mdl-25240856

RESUMO

Ind1, a mitochondrial P-loop NTPase is essential for assembly of respiratory complex-I. Respiratory complex-I (NADH: ubiquinone oxidoreductase), a large (mitochondrial inner membrane) enzyme, is made of 45 subunits and 8 iron-sulfur clusters. Ind1, an iron-sulfur cluster protein involved in the maturation of respiratory complex and binds an Fe/S cluster via a conserved CXXC motif in a labile way. Ind1 has been proposed as a specialized biogenesis factor involved in delivering the Fe/S clusters to the apo complex-I subunits. The IND1 gene is conserved in eukaryotes and is present in genomes of the species that retain functional respiratory complex-I. Depletion of human Ind1 causes ultra-structural changes in depleted mitochondria, including the loss of cristae membranes, massive remodeling of respiratory super complexes, and increased lactate production. Ind1 sequence bears known nucleotide binding domain motifs and was first classified as Nucleotide Binding Protein-Like (NUBPL). Despite the obvious importance of Ind1, very little is known about this protein; in particular its structure as well as its Fe/S cluster binding properties. In the present work we show that the expression of native huInd1 in Escherichia coli stimulates over-expression of the beta-lactamase TEM-1 from E. coli. The homology modeling of huInd1 shows hallmark of Rossmann fold, where a central beta sheet is covered by helices on either side. In the light of the modeled structure of huInd1, we hypothesize that huInd1 binds to the untranslated region (UTR) of the TEM-1 mRNA at 3' site and thereby reducing the possibility of its endonucleolytic cleavage, resulting in over-expression of TEM-1.


Assuntos
Resistência a Ampicilina , Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/metabolismo , beta-Lactamases/biossíntese , beta-Lactamases/isolamento & purificação , Escherichia coli/genética , Humanos , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Modelos Moleculares , Conformação Proteica , beta-Lactamases/genética
13.
Proc Natl Acad Sci U S A ; 108(12): 4811-6, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21383138

RESUMO

Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR-MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein-protein recognition needed for an efficient electron transfer process.


Assuntos
Redutases do Citocromo/química , Flavina-Adenina Dinucleotídeo/química , Proteínas de Transporte da Membrana Mitocondrial/química , Sítios de Ligação , Redutases do Citocromo/metabolismo , Transporte de Elétrons/fisiologia , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/química , Isoenzimas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Estrutura Terciária de Proteína , Especificidade por Substrato/fisiologia
14.
Protein Sci ; 33(3): e4910, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38358125

RESUMO

Fluorinated aromatic amino acids (FAAs) are promising tools when studying protein structure and dynamics by NMR spectroscopy. The incorporation FAAs in mammalian expression systems has been introduced only recently. Here, we investigate the effects of FAAs incorporation in proteins expressed in human cells, focusing on the probability of incorporation and its consequences on the 19 F NMR spectra. By combining 19 F NMR, direct MS and x-ray crystallography, we demonstrate that the probability of FAA incorporation is only a function of the FAA concentration in the expression medium and is a pure stochastic phenomenon. In contrast with the MS data, the x-ray structures of carbonic anhydrase II reveal that while the 3D structure is not affected, certain positions lack fluorine, suggesting that crystallization selectively excludes protein molecules featuring subtle conformational modifications. This study offers a predictive model of the FAA incorporation efficiency and provides a framework for controlling protein fluorination in mammalian expression systems.


Assuntos
Aminoácidos , Proteínas , Animais , Humanos , Aminoácidos/química , Proteínas/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Flúor/química , Mamíferos
15.
ACS Med Chem Lett ; 15(2): 250-257, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38352832

RESUMO

We have applied a proteolysis targeting chimera (PROTAC) technology to obtain a peptidomimetic molecule able to trigger the degradation of SARS-CoV-2 3-chymotrypsin-like protease (3CLPro). The PROTAC molecule was designed by conjugating a GC-376 based dipeptidyl 3CLPro ligand to a pomalidomide moiety through a piperazine-piperidine linker. NMR and crystallographic data complemented with enzymatic and cellular studies showed that (i) the dipeptidyl moiety of PROTAC binds to the active site of the dimeric state of SARS-CoV-2 3CLPro forming a reversible covalent bond with the sulfur atom of catalytic Cys145, (ii) the linker and the pomalidomide cereblon-ligand of PROTAC protrude from the protein, displaying a high degree of flexibility and no interactions with other regions of the protein, and (iii) PROTAC reduces the protein levels of SARS-CoV-2 3CLPro in cultured cells. This study paves the way for the future applicability of peptidomimetic PROTACs to tackle 3CLPro-dependent viral infections.

16.
J Am Chem Soc ; 134(3): 1442-5, 2012 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-22224850

RESUMO

The oxidative folding mechanism in the intermembrane space of human mitochondria underpins a disulfide relay system consisting of the import receptor Mia40 and the homodimeric FAD-dependent thiol oxidase ALR. The flavoprotein ALR receives two electrons per subunit from Mia40, which are then donated through one-electron reactions to two cytochrome c molecules, thus mediating a switch from two-electron to one-electron transfer. We dissect here the mechanism of the electron flux within ALR, characterizing at the atomic level the ALR intermediates that allow electrons to rapidly flow to cytochrome c. The intermediate critical for the electron-transfer process implies the formation of a specific inter-subunit disulfide which exclusively allows electron flow from Mia40 to FAD. This finding allows us to present a complete model for the electron-transfer pathway in ALR.


Assuntos
Redutases do Citocromo/metabolismo , Citocromos c/metabolismo , Dissulfetos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Dicroísmo Circular , Redutases do Citocromo/química , Citocromos c/química , Dissulfetos/química , Transporte de Elétrons , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Espectrofotometria Ultravioleta
17.
J Am Chem Soc ; 134(16): 7009-14, 2012 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-22471402

RESUMO

cis-Diamminedichloroplatinum(II) (cisplatin) is able to interact with human superoxide dismutase (hSOD1) in the disulfide oxidized apo form with a dissociation constant of 37 ± 3 µM through binding cysteine 111 (Cys111) located at the edge of the subunit interface. It also binds to Cu(2)-Zn(2) and Zn(2)-Zn(2) forms of hSOD1. Cisplatin inhibits aggregation of demetalated oxidized hSOD1, and it is further able to dissolve and monomerize oxidized hSOD1 oligomers in vitro and in cell, thus indicating its potential as a leading compound for amyotrophic lateral sclerosis.


Assuntos
Cisplatino/farmacologia , Inibidores Enzimáticos/farmacologia , Superóxido Dismutase/antagonistas & inibidores , Animais , Linhagem Celular , Cisplatino/química , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Soluções , Relação Estrutura-Atividade , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo
18.
Proc Natl Acad Sci U S A ; 106(17): 6980-5, 2009 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-19369197

RESUMO

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Multimerização Proteica , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Apoproteínas/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Superóxido Dismutase/genética , Superóxido Dismutase-1
19.
Biomolecules ; 12(7)2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35883565

RESUMO

The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron-sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation. The synthesis and distribution of Fe-S clusters are strictly controlled cellular processes that involve several mitochondrial proteins that specifically interact each other to form a complex machinery (Iron Sulfur Cluster assembly machinery, ISC machinery hereafter). This machinery ensures the correct assembly of both [2Fe-2S] and [4Fe-4S] clusters and their insertion in the mitochondrial target proteins. The present review provides a structural and molecular overview of the rare diseases associated with the genes encoding for the accessory proteins of the ISC machinery (i.e., GLRX5, ISCA1, ISCA2, IBA57, FDX2, BOLA3, IND1 and NFU1) involved in the assembly and insertion of [4Fe-4S] clusters in mitochondrial proteins. The disease-related missense mutations were mapped on the 3D structures of these accessory proteins or of their protein complexes, and the possible impact that these mutations have on their specific activity/function in the frame of the mitochondrial [4Fe-4S] protein biogenesis is described.


Assuntos
Proteínas Ferro-Enxofre , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Doenças Raras/metabolismo , Enxofre/metabolismo
20.
Microorganisms ; 10(4)2022 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-35456744

RESUMO

Microorganisms possess the potential to adapt to fluctuations in environmental parameters, and their evolution is driven by the continuous generation of mutations. The reversion of auxotrophic mutations has been widely studied; however, little is known about the reversion of frameshift mutations resulting in amino acid auxotrophy and on the structure and functioning of the protein encoded by the revertant mutated gene. The aims of this work were to analyze the appearance of reverse mutations over time and under different selective pressures and to investigate revertant enzymes' three-dimensional structures and their correlation with a different growth ability. Escherichia coli FB182 strain, carrying the hisF892 single nucleotide deletion resulting in histidine auxotrophy, was subjected to different selective pressures, and revertant mutants were isolated and characterized. The obtained results allowed us to identify different indels of different lengths located in different positions in the hisF gene, and relations with the incubation time and the selective pressure applied were observed. Moreover, the structure of the different mutant proteins was consistent with the respective revertant ability to grow in absence of histidine, highlighting a correlation between the mutations and the catalytic activity of the mutated HisF enzyme.

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