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1.
PLoS Genet ; 17(4): e1009539, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33914734

RESUMO

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.


Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP70/genética , Substituição de Aminoácidos/genética , Doença de Canavan/patologia , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética
2.
Biochem J ; 479(4): 479-501, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35089310

RESUMO

A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.


Assuntos
Fármacos Anti-HIV/farmacocinética , Farmacorresistência Viral/genética , Protease de HIV/genética , Substituição de Aminoácidos , Fator de Transcrição AraC/genética , Arabinose/metabolismo , Quimosina/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Fusão gag-pol/metabolismo , Produtos do Gene gag/metabolismo , Genes araC , Protease de HIV/química , Protease de HIV/isolamento & purificação , Protease de HIV/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saquinavir/antagonistas & inibidores , Saquinavir/farmacologia , Seleção Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato
3.
Arch Biochem Biophys ; 725: 109280, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35605676

RESUMO

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galß1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galß1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004-2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galß1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galß1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galß1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galß1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.


Assuntos
Bifidobacterium longum , Animais , Bifidobacterium longum/metabolismo , Bovinos , Fetuínas , Simulação de Acoplamento Molecular , Mucinas/metabolismo , Polissacarídeos/química , alfa-N-Acetilgalactosaminidase/química , alfa-N-Acetilgalactosaminidase/genética
4.
Biochemistry ; 60(45): 3398-3407, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34694774

RESUMO

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.


Assuntos
Mucina-1/química , Mucinas/química , alfa-N-Acetilgalactosaminidase/metabolismo , Bifidobacterium longum/metabolismo , Caseínas/metabolismo , Catálise , Hidrólise , Cinética , Mucina-1/metabolismo , Mucinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Água/química , alfa-N-Acetilgalactosaminidase/fisiologia
5.
J Am Chem Soc ; 143(36): 14540-14550, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34473923

RESUMO

Intrinsic disorder (ID) constitutes a new dimension to the protein structure-function relationship. The ability to undergo conformational changes upon binding is a key property of intrinsically disordered proteins and remains challenging to study using conventional methods. A 1994 paper by R. S. Spolar and M. T. Record presented a thermodynamic approach for estimating changes in conformational entropy based on heat capacity changes, allowing quantification of residues folding upon binding. Here, we adapt the method for studies of intrinsically disordered proteins. We integrate additional data to provide a broader experimental foundation for the underlying relations and, based on >500 protein-protein complexes involving disordered proteins, reassess a key relation between polar and nonpolar surface area changes, previously determined using globular protein folding. We demonstrate the improved suitability of the adapted method to studies of the folded αα-hub domain RST from radical-induced cell death 1, whose interactome is characterized by ID. From extensive thermodynamic data, quantifying the conformational entropy changes upon binding, and comparison to the NMR structure, the adapted method improves accuracy for ID-based studies. Furthermore, we apply the method, in conjunction with NMR, to reveal hitherto undetected effects of interaction-motif context. Thus, inclusion of the disordered context of the DREB2A RST-binding motif induces structuring of the binding motif, resulting in major enthalpy-entropy compensation in the interaction interface. This study, also evaluating additional interactions, demonstrates the strength of the ID-adapted Spolar-Record thermodynamic approach for dissection of structural features of ID-based interactions, easily overlooked in traditional studies, and for translation of these into mechanistic knowledge.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Entropia , Proteínas Intrinsicamente Desordenadas/química , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Fatores de Transcrição/química
6.
Anal Biochem ; 605: 113863, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738214

RESUMO

The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5-1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 µl is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 µg of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook.


Assuntos
Proteínas de Transporte/química , Peptídeos/química , Proteínas de Plantas/química , Desnaturação Proteica , Guanidina/química , Cinética , Conformação Proteica , Estabilidade Proteica , Termodinâmica , Ureia/química
7.
Cell Mol Life Sci ; 76(24): 4923-4943, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31134302

RESUMO

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.


Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação a DNA/química , DNA/química , Antígeno Nuclear de Célula em Proliferação/química , DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Espectroscopia de Ressonância Magnética , Antígeno Nuclear de Célula em Proliferação/genética , Conformação Proteica
8.
Biochemistry ; 57(41): 5949-5956, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30230312

RESUMO

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.


Assuntos
Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H3N2/química , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Prótons , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/química , Substituição de Aminoácidos , Humanos , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Canais Iônicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Mutação de Sentido Incorreto , Relação Estrutura-Atividade , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
9.
Biochemistry ; 57(41): 5957-5968, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30230310

RESUMO

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.


Assuntos
Antivirais , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Canais Iônicos , Mutação de Sentido Incorreto , Proteínas da Matriz Viral , Substituição de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Escherichia coli , Humanos , Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mutagênese , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
10.
J Biol Chem ; 292(2): 512-527, 2017 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-27881680

RESUMO

Intrinsically disordered protein regions (IDRs) lack a well defined three-dimensional structure but often facilitate key protein functions. Some interactions between IDRs and folded protein domains rely on short linear motifs (SLiMs). These motifs are challenging to identify, but once found they can point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein radical-induced cell death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound by RCD1, we analyzed the IDRs in three protein partners, DREB2A (dehydration-responsive element-binding protein 2A), ANAC013, and ANAC046, considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as (DE)X(1,2)(YF)X(1,4)(DE)L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher Kd values. Specifically, although the SLiM was surrounded by IDRs, individual intrinsic α-helix propensities varied as shown by CD spectroscopy. NMR spectroscopy further demonstrated that DREB2A underwent coupled folding and binding with α-helix formation upon interaction with RCD1, whereas peptides from ANAC013 and ANAC046 formed different structures or were fuzzy in the complexes. These findings allow us to present a model of the stress-associated RCD1-transcription factor interactome and to contribute to the emerging understanding of the interactions between folded hubs and their intrinsically disordered partners.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Modelos Moleculares , Proteínas Nucleares/química , Dobramento de Proteína , Fatores de Transcrição/química , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Biochem J ; 474(15): 2509-2532, 2017 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-28701416

RESUMO

Gene-specific transcription factors (TFs) are key regulatory components of signaling pathways, controlling, for example, cell growth, development, and stress responses. Their biological functions are determined by their molecular structures, as exemplified by their structured DNA-binding domains targeting specific cis-acting elements in genes, and by the significant lack of fixed tertiary structure in their extensive intrinsically disordered regions. Recent research in protein intrinsic disorder (ID) has changed our understanding of transcriptional activation domains from 'negative noodles' to ID regions with function-related, short sequence motifs and molecular recognition features with structural propensities. This review focuses on molecular aspects of TFs, which represent paradigms of ID-related features. Through specific examples, we review how the ID-associated flexibility of TFs enables them to participate in large interactomes, how they use only a few hydrophobic residues, short sequence motifs, prestructured motifs, and coupled folding and binding for their interactions with co-activators, and how their accessibility to post-translational modification affects their interactions. It is furthermore emphasized how classic biochemical concepts like allostery, conformational selection, induced fit, and feedback regulation are undergoing a revival with the appreciation of ID. The review also describes the most recent advances based on computational simulations of ID-based interaction mechanisms and structural analysis of ID in the context of full-length TFs and suggests future directions for research in TF ID.


Assuntos
Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Proteínas Intrinsicamente Desordenadas/química , Cinética , Processamento de Proteína Pós-Traducional , Termodinâmica , Fatores de Transcrição/química
12.
Mutagenesis ; 32(1): 23-31, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27470699

RESUMO

Nanofibrillated cellulose (NFC) is a sustainable and renewable nanomaterial, with diverse potential applications in the paper and medical industries. As NFC consists of long fibres of high aspect ratio, we examined here whether TEMPO-(2,2,6,6-tetramethyl-piperidin-1-oxyl) oxidised NFC (length 300-1000nm, thickness 10-25nm), administrated by a single pharyngeal aspiration, could be genotoxic to mice, locally in the lungs or systemically in the bone marrow. Female C57Bl/6 mice were treated with four different doses of NFC (10, 40, 80 and 200 µg/mouse), and samples were collected 24h later. DNA damage was assessed by the comet assay in bronchoalveolar lavage (BAL) and lung cells, and chromosome damage by the bone marrow erythrocyte micronucleus assay. Inflammation was evaluated by BAL cell counts and analysis of cytokines and histopathological alterations in the lungs. A significant induction of DNA damage was observed at the two lower doses of NFC in lung cells, whereas no increase was seen in BAL cells. No effect was detected in the bone marrow micronucleus assay, either. NFC increased the recruitment of inflammatory cells to the lungs, together with a dose-dependent increase in mRNA expression of tumour necrosis factor α, interleukins 1ß and 6, and chemokine (C-X-C motif) ligand 5, although there was no effect on the levels of the respective proteins. The histological analysis showed a dose-related accumulation of NFC in the bronchi, the alveoli and some in the cytoplasm of macrophages. In addition, neutrophilic accumulation in the alveolar lung space was observed with increasing dose. Our findings showed that NFC administered by pharyngeal aspiration caused an acute inflammatory response and DNA damage in the lungs, but no systemic genotoxic effect in the bone marrow. The present experimental design did not, however, allow us to determine whether the responses were transient or could persist for a longer time.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Celulose/toxicidade , Dano ao DNA , Pulmão/efeitos dos fármacos , Nanofibras/toxicidade , Animais , Células da Medula Óssea/metabolismo , Celulose/farmacologia , Ensaio Cometa , Citocinas , DNA/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Inflamação , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Micronúcleos com Defeito Cromossômico , Testes para Micronúcleos , Nanofibras/química
13.
Biochemistry ; 55(28): 3949-56, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27307139

RESUMO

Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced.


Assuntos
Aminoácidos/química , Proteínas de Bactérias/química , Cellulomonas , Dobramento de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Celulose/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Solubilidade
14.
Biochem J ; 465(2): 281-94, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25348421

RESUMO

Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas Nucleares , Fatores de Transcrição , Ativação Transcricional/fisiologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Biochemistry ; 54(11): 2032-9, 2015 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-25751413

RESUMO

Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the -1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in K(M) for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, K(A), for the divalent metal ion (Co²âº, Zn²âº, Mn²âº, or Cd²âº). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn²âº concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH <4.5; therefore, only the basic limb of the bell-shaped pH profile was analyzed.


Assuntos
Proteínas Arqueais/metabolismo , Cátions Bivalentes/metabolismo , Metais/metabolismo , Proteínas Mutantes/metabolismo , Sulfolobus solfataricus/enzimologia , alfa-Manosidase/metabolismo , Substituição de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cádmio/química , Cádmio/metabolismo , Domínio Catalítico , Cátions Bivalentes/química , Cobalto/química , Cobalto/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ligantes , Manganês/química , Manganês/metabolismo , Manosídeos/metabolismo , Metais/química , Proteínas Mutantes/química , Concentração Osmolar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Zinco/química , Zinco/metabolismo , alfa-Manosidase/química , alfa-Manosidase/genética
16.
Appl Environ Microbiol ; 81(10): 3395-404, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25746996

RESUMO

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.


Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/metabolismo , Citosina/metabolismo , DCMP Desaminase/metabolismo , Desoxirribonucleotídeos/metabolismo , Nucleotídeos de Desoxiuracil/biossíntese , Nucleotídeo Desaminases/metabolismo , Sequência de Aminoácidos , Bacillus/química , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vias Biossintéticas , Cristalografia por Raios X , DCMP Desaminase/química , DCMP Desaminase/genética , Cinética , Dados de Sequência Molecular , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/genética , Especificidade por Substrato
17.
Chem Res Toxicol ; 28(8): 1627-35, 2015 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-26208679

RESUMO

Nanofibrillar cellulose is a very promising innovation with diverse potential applications including high quality paper, coatings, and drug delivery carriers. The production of nanofibrillar cellulose on an industrial scale may lead to increased exposure to nanofibrillar cellulose both in the working environment and the general environment. Assessment of the potential health effects following exposure to nanofibrillar cellulose is therefore required. However, as nanofibrillar cellulose primarily consists of glucose moieties, detection of nanofibrillar cellulose in biological tissues is difficult. We have developed a simple and robust method for specific and sensitive detection of cellulose fibers, including nanofibrillar cellulose, in biological tissue, using a biotinylated carbohydrate binding module (CBM) of ß-1,4-glycanase (EXG:CBM) from the bacterium Cellulomonas fimi. EXG:CBM was expressed in Eschericia coli, purified, and biotinylated. EXG:CBM was shown to bind quantitatively to five different cellulose fibers including four different nanofibrillar celluloses. Biotinylated EXG:CBM was used to visualize cellulose fibers by either fluorescence- or horse radish peroxidase (HRP)-tagged avidin labeling. The HRP-EXG:CBM complex was used to visualize cellulose fibers in both cryopreserved and paraffin embedded lung tissue from mice dosed by pharyngeal aspiration with 10-200 µg/mouse. Detection was shown to be highly specific, and the assay appeared very robust. The present method represents a novel concept for the design of simple, robust, and highly specific detection methods for the detection of nanomaterials, which are otherwise difficult to visualize.


Assuntos
Celulose/metabolismo , Celulose/ultraestrutura , Glicosídeo Hidrolases/metabolismo , Nanofibras/ultraestrutura , Coloração e Rotulagem/métodos , Animais , Biotinilação , Glicosídeo Hidrolases/química , Imuno-Histoquímica , Pulmão/citologia , Camundongos , Ligação Proteica
18.
Arch Biochem Biophys ; 542: 28-38, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24262852

RESUMO

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.


Assuntos
Domínio Catalítico , Orotato Fosforribosiltransferase/química , Orotato Fosforribosiltransferase/metabolismo , Saccharomyces cerevisiae/enzimologia , Escherichia coli/genética , Cinética , Modelos Moleculares , Mutação , Orotato Fosforribosiltransferase/genética , Orotato Fosforribosiltransferase/isolamento & purificação , Fosforribosil Pirofosfato/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genética , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/metabolismo
19.
ACS Synth Biol ; 13(3): 862-875, 2024 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-38357862

RESUMO

Enzymes are indispensable biocatalysts for numerous industrial applications, yet stability, selectivity, and restricted substrate recognition present limitations for their use. Despite the importance of enzyme engineering in overcoming these limitations, success is often challenged by the intricate architecture of enzymes derived from natural sources. Recent advances in computational methods have enabled the de novo design of simplified scaffolds with specific functional sites. Such scaffolds may be advantageous as platforms for enzyme engineering. Here, we present a strategy for the de novo design of a simplified scaffold of an endo-α-N-acetylgalactosaminidase active site, a glycoside hydrolase from the GH101 enzyme family. Using a combination of trRosetta hallucination, iterative cycles of deep-learning-based structure prediction, and ProteinMPNN sequence design, we designed proteins with 290 amino acids incorporating the active site while reducing the molecular weight by over 100 kDa compared to the initial endo-α-N-acetylgalactosaminidase. Of 11 tested designs, six were expressed as soluble monomers, displaying similar or increased thermostabilities compared to the natural enzyme. Despite lacking detectable enzymatic activity, the experimentally determined crystal structures of a representative design closely matched the design with a root-mean-square deviation of 1.0 Å, with most catalytically important side chains within 2.0 Å. The results highlight the potential of scaffold hallucination in designing proteins that may serve as a foundation for subsequent enzyme engineering.


Assuntos
Proteínas de Bactérias , Glicosídeo Hidrolases , Domínio Catalítico , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , alfa-N-Acetilgalactosaminidase/química , alfa-N-Acetilgalactosaminidase/metabolismo , Proteínas de Bactérias/metabolismo , Especificidade por Substrato
20.
Biochemistry ; 51(40): 8039-46, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-22989181

RESUMO

The active site for the family GH38 class II α-mannosidase is constituted in part by a divalent metal ion, mostly Zn(2+), as revealed in the crystal structures of enzymes from both animal and bacterial sources. The metal ion coordinates to the bound substrate and side chains of conserved amino acid residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent metal ion on the associated steady-state kinetic parameters, K(M) and k(cat), for various substrates. With p-nitrophenyl-α-d-mannoside as a substrate, the enzyme showed activity in the presence of Co(2+), Cd(2+), Mn(2+), and Zn(2+), whereas Ni(2+) and Cu(2+) were inhibitory and nonactivating. Co(2+) was the preferred metal ion, with a k(cat)/K(M) value of about 120 mM(-1) s(-1), 6 times higher than that with Cd(2+) and Zn(2+) and 10 times higher than that with Mn(2+). With α-1,2-, α-1,3-, α-1,4-, or α-1,6-mannobiose as a substrate, Co(2+) was the only metal ion promoting hydrolysis of all substrates; however, Mn(2+), Cd(2+), and Zn(2+) could substitute to a varying extent. A change in the divalent metal ion generally affected the K(M) for the hydrolysis of p-nitrophenyl-α-d-mannoside; however, changes in both k(cat) and K(M) for the hydrolysis of α-mannobioses were observed, along with changing preferences for the glycosidic linkage. Finally, it was found that the metal ion and substrate bind in that order via a steady-state, ordered, sequential mechanism.


Assuntos
Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Metais/metabolismo , Sulfolobus solfataricus/enzimologia , alfa-Manosidase/classificação , alfa-Manosidase/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Metais/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Especificidade por Substrato , alfa-Manosidase/genética
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