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2.
Curr Top Med Chem ; 19(6): 467-485, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31184298

RESUMO

BACKGROUND: Kinases are key modulators in regulating diverse range of cellular activities and are an essential part of the protein-protein interactome. Understanding the interaction of kinases with different substrates and other proteins is vital to decode the cell signaling machinery as well as causative mechanism for disease onset and progression. OBJECTIVE: The objective of this review is to present all studies on the structure and function of few important kinases and highlight the protein-protein interaction (PPI) mechanism of kinases and the kinase specific interactome databases and how such studies could be utilized to develop anticancer drugs. METHODS: The article is a review of the detailed description of the various domains in kinases that are involved in protein-protein interactions and specific inhibitors developed targeting these PPI domains. RESULTS: The review has surfaced in depth the interacting domains in key kinases and their features and the roles of PPI in the human kinome and the various signaling cascades that are involved in certain types of cancer. CONCLUSION: The insight availed into the mechanism of existing peptide inhibitors and peptidomimetics against kinases will pave way for the design and generation of domain specific peptide inhibitors with better productivity and efficiency and the various software and servers available can be of great use for the identification and analysis of protein-protein interactions.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Antineoplásicos/química , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Peptidomiméticos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/química
3.
Microbiol Res ; 223-225: 22-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178048

RESUMO

The Deinococcus radiodurans multipartite genome consists of 2 chromosomes and 2 plasmids Its genome encodes 4 ParA and 4 ParB proteins on different replicons. Multiple sequence alignments of ParBs encoded on these genome elements showed that ParB of primary chromosome (ParB1) is close to chromosomal type ParB and is found to be different from ParBs encoded on chromosome II (ParB2) and megaplasmid (ParB3) elements. We observed that ParB1, ParB2 and ParB3 exist as dimer in solution and these proteins interact to self but not to its homologs in D. radiodurans, suggesting the specificity in ParBs dimerization. The parB1 deletion mutant showed slow growth under normal condition and relatively reduced resistance to γ-radiation as compared to wild type. The parB2 and parB3 mutants maintained without selection pressure showed loss of radioresistance, which was not observed when maintained with selection pressure. Nearly half of the populations of these mutants showed resistance to antibiotics marked to respective genome elements. Interestingly, all the parB mutants showed increased copy numbers of cognate genome element in cells maintained with antibiotics possibly due to arrest in genome segregation. These results suggested that ParB proteins encoded on multipartite genome system in D. radiodurans form homodimer and not heterodimer with other ParB homologs, and they independently regulate the segregation of respective genome elements. The roles of ParB1 proteins in normal as well as radiation stressed growth of this bacterium have also been ascertained.


Assuntos
Proteínas de Bactérias/genética , Deinococcus/genética , Genes Bacterianos/genética , Sequência de Aminoácidos , Cromossomos Bacterianos/genética , Clonagem Molecular , Deinococcus/efeitos da radiação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Deleção de Sequência
4.
Nat Commun ; 10(1): 2636, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201318

RESUMO

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Modelos Moleculares , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Anticorpos de Domínio Único/metabolismo
5.
Microbiol Immunol ; 63(7): 285-288, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31166044

RESUMO

Phosphatidylinositol-4 kinase III ß (PI4KB) is a host factor that is required for enterovirus (EV) replication. In this study, the importance of host proteins that interact with PI4KB in EV replication was analyzed by trans complementation with PI4KB mutants in a PI4KB-knockout cell line. Ectopically expressed PI4KB mutants, which lack binding regions for ACBD3, RAB11, and 14-3-3 proteins, rescued replication of poliovirus and enterovirus 71. These findings suggest that interaction of PI4KB with these host proteins is not essential for EV replication once PI4KB has been expressed and that PI4KB is functionally independent from these host proteins regarding EV replication.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Enterovirus/metabolismo , Domínios e Motivos de Interação entre Proteínas , Replicação Viral/fisiologia , 1-Fosfatidilinositol 4-Quinase/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Linhagem Celular , Infecções por Enterovirus , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Poliovirus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
6.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 564-577, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205019

RESUMO

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.


Assuntos
Proteínas de Bactérias/química , N-Acilneuraminato Citidililtransferase/química , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X/métodos , Cistina Difosfato/química , Cistina Difosfato/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferase/farmacologia , N-Acilneuraminato Citidililtransferase/fisiologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Ácidos Siálicos/metabolismo
7.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 578-591, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205020

RESUMO

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (ßFXIIaHis) with yields of ∼1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-ßFXIIaHis). Crystal structures were determined of MBP-ßFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of ßFXIIaHis in isolation. The ßFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the ßFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with ßFXIIa are also described. These structural studies of ßFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.


Assuntos
Fator XIIa , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão/química , Clorometilcetonas de Aminoácidos/química , Animais , Sítios de Ligação , Linhagem Celular , Cristalização , Cristalografia por Raios X/métodos , Drosophila melanogaster , Fator XIIa/química , Fator XIIa/metabolismo , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato
8.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 592-604, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205021

RESUMO

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5-2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.


Assuntos
Homocisteína/análogos & derivados , Monoéster Fosfórico Hidrolases/química , Fosfosserina/química , Serina/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X/métodos , Escherichia coli , Homocisteína/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Fosfosserina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Serina/metabolismo
9.
Biochimie ; 163: 117-127, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31194995

RESUMO

Protein phosphorylation is one of the main mechanisms by which signals are transmitted in eukaryotic cells, and it plays a crucial regulatory role in almost all cellular processes. In yeast, more than half of the proteins are phosphorylated in at least one site, and over 20,000 phosphopeptides have been experimentally verified. However, the functional consequences of these phosphorylation events for most of the identified phosphosites are unknown. A family of protein interaction domains selectively recognises phosphorylated motifs to recruit regulatory proteins and activate signalling pathways. Nine classes of dedicated modules are coded by the yeast genome: 14-3-3, FHA, WD40, BRCT, WW, PBD, and SH2. The recognition specificity relies on a few residues on the target protein and has coevolved with kinase specificity. In the present study, we review the current knowledge concerning yeast phospho-binding domains and their networks. We emphasise the relevance of both positive and negative amino acid selection to orchestrate the highly regulated outcomes of inter- and intra-molecular interactions. Finally, we hypothesise that only a small fraction of yeast phosphorylation events leads to the creation of a docking site on the target molecule, while many have a direct effect on the protein or, as has been proposed, have no function at all.


Assuntos
Fosfopeptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Proteínas de Saccharomyces cerevisiae/fisiologia
10.
Nat Methods ; 16(6): 493-496, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110281

RESUMO

Here, we introduce fluorescence intensity fluctuation spectrometry for determining the identity, abundance and stability of protein oligomers. This approach was tested on monomers and oligomers of known sizes and was used to uncover the oligomeric states of the epidermal growth factor receptor and the secretin receptor in the presence and absence of their agonist ligands. This method is fast and is scalable for high-throughput screening of drugs targeting protein-protein interactions.


Assuntos
Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas-G/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Ligantes , Microscopia Confocal , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Espectrometria de Fluorescência
11.
Microb Pathog ; 132: 141-149, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31051192

RESUMO

The plants resist/tolerate unfavorable conditions in their natural habitats by using different but aligned and integrated defense mechanisms. Such defense responses include not only morphological and physiological adaptations but also the genomic and transcriptomic reconfiguration. Microbial attack on plants activates multiple pro-survival pathways such as transcriptional reprogramming, hypersensitive response (HR), antioxidant defense system and metabolic remodeling. Up-regulation of these processes during biotic stress conditions directly relates with plant survival. Over the years, hundreds of plant transcription factors (TFs) belonging to diverse families have been identified. Zinc finger protein (ZFP) TFs have crucial role in phytohormone response, plant growth and development, stress tolerance, transcriptional regulation, RNA binding and protein-protein interactions. Recent research progress has revealed regulatory and biological functions of ZFPs in incrementing plant resistance to pathogens. Integration of transcriptional activity with metabolic modulations has miniaturized plant innate immunity. However, the precise roles of different zinc finger TFs in plant immunity to pathogens have not been thoroughly analyzed. This review consolidates the pivotal functioning of zinc finger TFs and proposes the integrative understanding as foundation for the plant growth and development including the stress responses.


Assuntos
Anti-Infecciosos/farmacologia , Imunidade Vegetal , Fatores de Transcrição/fisiologia , Dedos de Zinco/fisiologia , Adaptação Fisiológica , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Imunidade Inata , Filogenia , Desenvolvimento Vegetal , Reguladores de Crescimento de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas com Motivo de Reconhecimento de RNA , Estresse Fisiológico
12.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 324-331, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045561

RESUMO

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.


Assuntos
Proteínas de Bactérias/química , Hidrocarbonetos Halogenados/química , Hidrolases/química , Psychrobacter/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hidrocarbonetos Halogenados/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Psychrobacter/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica
13.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 332-339, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045562

RESUMO

SUMOylation is a post-translational modification in which a small ubiquitin-like molecule (SUMO) is appended to substrate proteins and is known to influence myriads of biological processes. A delicate interplay between several families of SUMOylation proteins and their substrates ensures the proper level of SUMOylation required for normal cell function. Among the SUMO proteins, SUMO2 is known to form mono-SUMOylated proteins and engage in poly-SUMO chain formation, while sentrin-specific protease 1 (SENP1) is a key enzyme in regulating both events. Determination of the SENP1-SUMO2 interaction is therefore necessary to better understand SUMOylation. In this regard, the current paper reports the noncovalent structure of SENP1 in complex with SUMO2, which was refined to a resolution of 2.62 Šwith R and Rfree values of 22.92% and 27.66%, respectively. The structure shows that SENP1-SUMO2 complex formation is driven largely by polar interactions and limited hydrophobic contacts. The essential C-terminal motif (QQTGG) of SUMO2 is stabilized by a number of specific bonding interactions that enable it to protrude into the catalytic triad of SENP1 and provide the arrangement necessary for maturation of SUMO and deSUMOylation activity. Overall, the structure shows a number of structural details that pinpoint the basis of SENP1-SUMO2 complex formation.


Assuntos
Cisteína Endopeptidases/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Homologia Estrutural de Proteína , Termodinâmica
14.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 340-347, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045563

RESUMO

Ebola virus is an emerging virus that is capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome are carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Šresolution single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution, while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.


Assuntos
Ebolavirus/química , Nucleocapsídeo/química , Nucleoproteínas/química , RNA Viral/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleocapsídeo/ultraestrutura , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 348-358, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045564

RESUMO

Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1-H6 and H7-H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepTSo2 from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepTSo2-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepTSo2 were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepTSo2 incorporated into Salipro nanoparticles at 4.1 Šresolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepTSo2 to be determined at resolutions of 3.5 and 3.9 Å. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepTSo2, in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Shewanella/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Shewanella/metabolismo , Especificidade por Substrato
16.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 359-367, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045565

RESUMO

As of 2017, tuberculosis had infected 1.7 billion people (23% of the population of the world) and caused ten million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multidrug resistant. Thus, the identification of novel druggable targets is essential to combat the proliferation of these drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ) is a key protein involved in cytokinesis, an important process for Mtb proliferation and viability. FtsZ is required for bacterial cell division because it polymerizes into a structure called the Z-ring, which recruits accessory division proteins to the septum. Here, the crystal structure of the MtbFtsZ protein has been determined to 3.46 Šresolution and is described as a dimer of trimers, with an inter-subunit interface between protomers AB and DE. In this work, a novel conformation of MtbFtsZ is revealed involving the T9 loop and the nucleotide-binding pocket of protomers BC and EF.


Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Mycobacterium tuberculosis/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Divisão Celular , Clonagem Molecular , Cristalografia por Raios X , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Temperatura Ambiente
17.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 368-376, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045566

RESUMO

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Šand was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Šresolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Cloretos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Haemophilus influenzae/química , Lipoproteínas/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína
18.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 377-384, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045567

RESUMO

With better tools for data processing and with synchrotron beamlines that are capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the `first-choice' method for de novo protein structure determination. However, for many proteins native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5 MDa calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure had been determined. Given that Ca atoms will be present in a significant percentage of proteins that remain uncharacterized, many aspects of the data-collection and processing methods described here could be broadly applied for routine de novo structure elucidation.


Assuntos
Adesinas Bacterianas/química , Proteínas de Bactérias/química , Cálcio/química , Gelo/análise , Marinomonas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Organismos Aquáticos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios X
19.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 392-396, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045569

RESUMO

Grx1, a cytosolic thiol-disulfide oxidoreductase, actively maintains cellular redox homeostasis using glutathione substrates (reduced, GSH, and oxidized, GSSG). Here, the crystallization of reduced Grx1 from the yeast Saccharomyces cerevisiae (yGrx1) in space group P212121 and its structure solution and refinement to 1.22 Šresolution are reported. To study the structure-function relationship of yeast Grx1, the crystal structure of reduced yGrx1 was compared with the existing structures of the oxidized and glutathionylated forms. These comparisons revealed structural differences in the conformations of residues neighbouring the Cys27-Cys30 active site which accompany alterations in the redox status of the protein.


Assuntos
Cisteína/química , Glutarredoxinas/química , Glutationa/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
20.
Anal Bioanal Chem ; 411(17): 3721-3729, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139859

RESUMO

Combining mass spectrometry (MS) with surface plasmon resonance (SPR) makes it possible to identify the chemical structures of the interacting molecules studied by SPR. Different approaches for coupling surface plasmon resonance sensors to mass spectrometry were developed. This article aims to summarize the established approaches and their applications to study biomolecular interactions. Three representative interactions were reviewed: protein-protein interactions, enzyme-substrate/inhibitor interactions, and protein-small molecule interactions.


Assuntos
Espectrometria de Massas/métodos , Ressonância de Plasmônio de Superfície/métodos , Enzimas/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Especificidade por Substrato
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