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1.
J Struct Biol ; 213(2): 107715, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33705979

RESUMO

The 106-residue protein Q4DY78 (UniProt accession number) from Trypanosoma cruzi is highly conserved in the related kinetoplastid pathogens Trypanosoma brucei and Leishmania major. Given the essentiality of its orthologue in T. brucei, the high sequence conservation with other trypanosomatid proteins, and the low sequence similarity with mammalian proteins, Q4DY78 is an attractive protein for structural characterization. Here, we solved the structure of Q4DY78 by solution NMR and evaluated its backbone dynamics. Q4DY78 is composed of five α -helices and a small, two-stranded antiparallel ß-sheet. The backbone RMSD is 0.22 ± 0.05 Å for the representative ensemble of the 20 lowest-energy structures. Q4DY78 is overall rigid, except for N-terminal residues (V8 to I10), residues at loop 4 (K57 to G65) and residues at the C-terminus (F89 to F112). Q4DY78 has a short motif FPCAP that could potentially mediate interactions with the host cytoskeleton via interaction with EVH1 (Drosophila Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1) domains. Albeit Q4DY78 lacks calcium-binding motifs, its fold resembles that of eukaryotic calcium-binding proteins such as calcitracin, calmodulin, and polcacin Bet V4. We characterized this novel protein with a calcium binding fold without the capacity to bind calcium.


Assuntos
Proteínas de Protozoários/química , Trypanosoma cruzi/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Moléculas de Adesão Celular/química , Dicroísmo Circular , Sequência Conservada , Motivos EF Hand , Proteínas dos Microfilamentos/química , Modelos Moleculares , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Conformação Proteica em alfa-Hélice , Estrutura Secundária de Proteína , Proteínas de Protozoários/metabolismo
2.
J Struct Biol ; 211(2): 107536, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32473201

RESUMO

Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ßßαßßαßß topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction.


Assuntos
Proteínas de Protozoários/ultraestrutura , Trypanosoma cruzi/ultraestrutura , Animais , Humanos , Leishmania major/ultraestrutura , Modelos Moleculares , Proteínas de Protozoários/genética , Espalhamento a Baixo Ângulo , Trypanosoma brucei brucei/ultraestrutura , Trypanosoma cruzi/genética , Difração de Raios X
3.
Biomol NMR Assign ; 14(1): 119-122, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32030620

RESUMO

Tuberculosis is one of the deadliest diseases worldwide affecting approximately 10 million people in 2018. This classifies tuberculosis as epidemic in several countries and leads to an increasing number of multidrug-resistant strains. Thus, the development of new drugs is essential to effective treatments. A potential drug target is the ribose-5-phosphate isomerase, a ubiquitous enzyme important to nucleotide and cofactor biosynthesis. Here, we report the backbone assignment of ribose-5-phosphate isomerase of Mycobacterium tuberculosis (MtRpiB) that has been performed by triple resonance sequential approach using a [13C, 15N, 2H]-labeled protein. This is the first ribose-5-phosphate isomerase, an enzyme previously classified as highly druggable, to be assigned. These data will be important to further screening studies to find inhibitors and determine their interaction with MtRpiB.


Assuntos
Aldose-Cetose Isomerases/química , Mycobacterium tuberculosis/enzimologia , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína
4.
Biomol NMR Assign ; 13(1): 239-243, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30879170

RESUMO

FK506 Binding Proteins (FKBPs) are a family of highly conserved and important proteins that possess a peptidyl cis-trans isomerase (PPIases) domain. Human FKBP12 is a prototype of this family and it is involved in many diseases due to its interaction with the immunosuppressive drugs FK506 and rapamycin. They inhibit calcineurin and mTOR complex, respectively, leading to parasite death by inhibiting cell proliferation through cytokinesis blockade being an important target to find new drugs. Tuberculosis is a disease that causes important impacts on public health worldwide. In this context, MtFKBP12 is a putative peptidyl prolyl cis-trans isomerase from Mycobacterium tuberculosis and here we report the NMR chemical shift assignment for 1H, 15N and 13C nuclei in the backbone and side chains of the MtFKBP12. This lays the foundation for further structural studies, backbone dynamics, mapping of interactions and drug screening and development. We have found through the NMR spectrum that the protein is well folded with narrow peaks and almost none overlap in 15N-HSQC. Prediction of secondary structure using Talos-N server showed great similarity with other proteins from this family.


Assuntos
Mycobacterium tuberculosis/enzimologia , Ressonância Magnética Nuclear Biomolecular , Proteína 1A de Ligação a Tacrolimo/química , Isótopos de Carbono , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Prótons
5.
Biochimie ; 150: 37-47, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29730302

RESUMO

Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI-carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously blocks these two mechanistic classes of enzymes.


Assuntos
Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/metabolismo , Poliquetos/enzimologia , Inibidores de Proteases/química , Serina Proteases/metabolismo , Animais , Cinética , Espectroscopia de Ressonância Magnética , Tripsina/química , Tripsina/metabolismo
6.
Biomol NMR Assign ; 10(2): 325-8, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27356988

RESUMO

Trypanosoma cruzi, Trypanosma brucei and Leishmania spp. are kinetoplastid protozoa causative agents of Chagas disease, sleeping sickness and leishmaniasis, respectively, neglected tropical diseases estimated to infect millions of people worldwide. Their genome sequencing has revealed approximately 50 % of genes encoding hypothetical proteins of unknown function, opening possibilities for novel target identification and drug discovery. Q4DY78 is a putative essential protein from T. cruzi conserved in the related kinetoplastids and divergent from mammalian host proteins. Here we report the (1)H, (15)N, and (13)C chemical shift assignments and secondary structure analysis of the Q4DY78 protein as basis for NMR structure determination, functional analysis and drug screening.


Assuntos
Sequência Conservada , Ressonância Magnética Nuclear Biomolecular , Proteínas de Protozoários/química , Trypanosoma cruzi , Estrutura Secundária de Proteína
7.
Biomol NMR Assign ; 10(1): 153-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26547437

RESUMO

A protease inhibitor (CmPI-II) (UNIPROT: IPK2_CENMR) from the marine mollusc Cenchritis muricatus, has been isolated and characterized. It is the first member of a new group (group 3) of non-classical Kazal-type inhibitors. CmPI-II is a tight-binding inhibitor of serine proteases: trypsin, human neutrophil elastase (HNE), subtilisin A and pancreatic elastase. This specificity is exceptional in the members of Kazal-type inhibitor family. Several models of three-dimensional structure of CmPI-II have been constructed by homology with other inhibitors of the family but its structure has not yet been solved experimentally. Here we report the (1)H, (15)N and (13)C chemical shift assignments of CmPI-II as basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified three ß-strands ß1: residues 14-19, ß2: 23-35 and ß3: 43-45 and one helix α1: 28-37 arranged in the sequential order ß1-ß2-α1-ß3. These secondary structure elements suggest that CmPI-II adopts the typical scaffold of a Kazal-type inhibitor.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Inibidores de Serina Proteinase/química , Caramujos , Animais , Estrutura Secundária de Proteína
8.
J Pharm Sci ; 105(9): 2648-2655, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26372719

RESUMO

The experiments of carvedilol form II, form III, and hydrate by (13)C and (15)N cross-polarization magic-angle spinning (CP MAS) are reported. The GIPAW (gauge-including projector-augmented wave) method from DFT (density functional theory) calculations was used to simulate (13)C and (15)N chemical shifts. A very good agreement was found for the comparison between the global results of experimental and calculated nuclear magnetic resonance (NMR) chemical shifts for carvedilol polymorphs. This work aims a comprehensive understanding of carvedilol crystalline forms employing solution and solid-state NMR as well as DFT calculations.


Assuntos
Carbazóis/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Propanolaminas/química , Isótopos de Carbono/química , Carvedilol , Cristalização , Cristalografia por Raios X , Estrutura Molecular , Isótopos de Nitrogênio/química
9.
Proteins ; 82(6): 1022-34, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24218049

RESUMO

Sticholysin I (StI), an actinoporin expressed as a water-soluble protein by the sea anemone Stichodactyla helianthus, binds to natural and model membranes, forming oligomeric pores. It is proposed that the first event of a multistep pore formation mechanism consists of the monomeric protein attachment to the lipid bilayer. To date there is no high-resolution structure of the actinoporin pore or other membrane-bound form available. Here we evaluated StI:micelle complexes of variable lipid composition to look for a suitable model for NMR studies. Micelles of pure or mixed lysophospholipids and of dihexanoyl phosphatidylcholine (DHPC) were examined. The StI:DHPC micelle was found to be the best system, yielding a stable sample and good quality spectra. A comprehensive chemical shift perturbation analysis was performed to map the StI membrane recognition site in the presence of DHPC micelles. The region mapped (residues F(51), R(52), S(53) in loop 3; F(107), D(108), Y(109), W(111), Y(112), W(115) in loop 7; Q(129), Y(132), D(134), M(135), Y(136), Y(137), G(138) in helix-α2) is in agreement with previously reported data, but additional residues were found to interact, especially residues V(81), A(82), T(83), G(84) in loop 5, and A(85), A(87) in strand-ß5. Backbone dynamics measurements of StI free in solution and bound to micelles highlighted the relevance of protein flexibility for membrane binding and suggested that a conformer selection process may take place during protein-membrane interaction. We conclude that the StI:DHPC micelles system is a suitable model for further characterization of an actinoporin membrane-bound form by solution NMR.


Assuntos
Éteres Fosfolipídicos/química , Animais , Membrana Celular/química , Micelas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Compostos Orgânicos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Anêmonas-do-Mar , Soluções , Propriedades de Superfície
10.
Protein Expr Purif ; 48(2): 253-60, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16584890

RESUMO

Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with (15)N and (13)C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general.


Assuntos
Endotelina-1/biossíntese , Endotelina-1/isolamento & purificação , Expressão Gênica , Ressonância Magnética Nuclear Biomolecular , Eletroforese em Gel de Poliacrilamida , Endotelina-1/genética , Escherichia coli/genética , Humanos , Marcação por Isótopo , Isótopos de Nitrogênio , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
FEBS Lett ; 579(17): 3534-8, 2005 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-15961083

RESUMO

Phox and Bem1 (PB1) domains mediate protein-protein interactions via the formation of homo- or hetero-dimers. The C-terminal PB1 domain of yeast cell division cycle 24 (CDC24p), a guanine-nucleotide exchange factor involved in cell polarity establishment, is known to interact with the PB1 domain occurring in bud emergence MSB1 interacting 1 (BEM1p) during the regulation of the yeast budding process via its OPR/PC/AID (OPCA) motif. Here, we present the structure of an N-terminally truncated version of the Sc CDC24p PB1 domain. It shows a different topology of the beta-sheet than the long form. However, the C-terminal part of the structure shows the conserved PB1 domain features including the OPCA motif with a slight rearrangement of helix alpha1. Residues which are important for the heterodimerization with BEM1p are structurally preserved.


Assuntos
Proteínas de Ciclo Celular/química , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Soluções/química
12.
J Mol Biol ; 348(2): 399-408, 2005 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15811376

RESUMO

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Fetais/química , Proteínas Fetais/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Mapeamento de Epitopos , Forminas , Humanos , Ligantes , Proteínas dos Microfilamentos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato , Termodinâmica
13.
J Immunol ; 174(2): 942-52, 2005 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-15634917

RESUMO

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.


Assuntos
Antígenos de Neoplasias/imunologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Biomarcadores Tumorais/imunologia , Moléculas de Adesão Celular/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/normas , Animais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/isolamento & purificação , Autoanticorpos/biossíntese , Autoantígenos/isolamento & purificação , Baculoviridae/genética , Baculoviridae/imunologia , Biomarcadores Tumorais/isolamento & purificação , Biomarcadores Tumorais/normas , Neoplasias da Mama/imunologia , Moléculas de Adesão Celular/isolamento & purificação , Moléculas de Adesão Celular/normas , Linhagem Celular , Neoplasias Colorretais/imunologia , Relação Dose-Resposta Imunológica , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Contaminação de Medicamentos , Molécula de Adesão da Célula Epitelial , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Desnaturação Proteica , Dobramento de Proteína , Controle de Qualidade , Proteínas Recombinantes/isolamento & purificação , Neoplasias Gástricas/imunologia
14.
Protein Sci ; 12(3): 491-500, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12592019

RESUMO

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dipeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Dipeptídeos/química , Humanos , Cinética , Ligantes , Cadeias de Markov , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Biblioteca de Peptídeos , Fosfotirosina , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos
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