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1.
Nucleic Acids Res ; 45(17): 10293-10305, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973465

RESUMO

Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/ß domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
2.
Elife ; 62017 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-28792888

RESUMO

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos
3.
Nat Commun ; 8: 15424, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28548080

RESUMO

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.


Assuntos
Antineoplásicos/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cristalografia por Raios X , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferons/imunologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Biol Chem ; 290(41): 24975-85, 2015 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-26286748

RESUMO

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.


Assuntos
Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Biocatálise , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dano ao DNA , Ativação Enzimática/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fator de Crescimento Insulin-Like I/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/química , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Treonina/metabolismo
5.
J Am Chem Soc ; 137(20): 6506-16, 2015 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-25924808

RESUMO

The integration of atomic-resolution experimental and computational methods offers the potential for elucidating key aspects of protein folding that are not revealed by either approach alone. Here, we combine equilibrium NMR measurements of thermal unfolding and long molecular dynamics simulations to investigate the folding of gpW, a protein with two-state-like, fast folding dynamics and cooperative equilibrium unfolding behavior. Experiments and simulations expose a remarkably complex pattern of structural changes that occur at the atomic level and from which the detailed network of residue-residue couplings associated with cooperative folding emerges. Such thermodynamic residue-residue couplings appear to be linked to the order of mechanistically significant events that take place during the folding process. Our results on gpW indicate that the methods employed in this study are likely to prove broadly applicable to the fine analysis of folding mechanisms in fast folding proteins.


Assuntos
Simulação de Dinâmica Molecular , Dobramento de Proteína , Proteínas/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas/metabolismo , Termodinâmica , Fatores de Tempo
6.
Structure ; 21(10): 1800-11, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-23994011

RESUMO

The seven C-terminal CCCH-type zinc fingers of Nab2p bind the poly(A) tail of mRNA (∼A25). Using NMR, we demonstrated that the first four (Zf1-Zf4) contain two structurally independent tandems (TZF12 and TZF34) and bind A12 with moderate affinity (KD = 2.3 µM). Nab2p TZF12 contains a long α helix that contacts the zinc fingers Zf1 and Zf2 to arrange them similarly to Zf6-7 in the Nab2p Zf5-7 structure. Nab2p TZF34 exhibits a distinctive two-fold symmetry of the zinc centers with mutual recognition of histidine ligands. Our mutagenesis and NMR data demonstrate that the α helix of TZF12 and Zf3 of TZF34 define the RNA-binding interface, while Zf1, Zf2, and Zf4 seem to be excluded. These results further our understanding of polyadenosine RNA recognition by the CCCH domain of Nab2p. Moreover, we describe a hypothetical mechanism for controlling poly(A) tail length with specific roles for TZF12, TZF34, and Zf5-7 domains.


Assuntos
Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Sequência Conservada , Complexos de Coordenação/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia Estrutural de Proteína , Termodinâmica , Dedos de Zinco
8.
J Mol Recognit ; 25(12): 665-73, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23192964

RESUMO

According to biochemical assays, the Bcl-2 protein Diva from mouse regulates programmed cell death by heterodimerizing with other members of the family and by interacting with the apoptotic protease-activating factor Apaf-1. In typical Bcl-2 heterodimers, peptide fragments comprising the Bcl-2 homology domain 3 (BH3 domain) of proapoptotic members are capable of forming functional complexes with prosurvival proteins. High-resolution structural studies have revealed that the BH3 peptide forms an α-helix positioned in a canonical hydrophobic cleft of the antiapoptotic protein. Because Diva shows mutations in conserved residues within this area, it has been proposed to have a different interacting surface. However, we showed previously that Diva binds through the canonical groove the BH3 peptide of the human Bcl-2 killing member Harakiri. To further test Diva's binding capabilities, here we show Nuclear Magnetic Resonance (NMR) data, indicating that Diva binds peptides derived from the BH3 domain of several other proapoptotic Bcl-2 proteins, including mouse Harakiri, Bid, Bak and Bmf. We have measured the binding affinities of the heterodimers, which show significant variability. Structural models of the protein-peptide complexes based on NMR chemical shift perturbation data indicate that the binding surface is analogous. These models do not rely on NMR NOE (Nuclear Overhauser Effect) data, and thus our results can only suggest that the complexes share similar intermolecular interactions. However, the observed affinity differences correlate with the α-helical population of the BH3-peptides obtained from circular dichroism experiments, which highlights a role of conformational selection in the binding mechanism. Altogether, our results shed light on important factors governing Diva-BH3 peptide molecular recognition mode.


Assuntos
Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
9.
Bioorg Med Chem Lett ; 22(1): 444-8, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22119467

RESUMO

Based on ß-turn-like BDNF loops 2 and 4, involved in receptor interaction, cyclic peptide replicas were designed, synthesized and tested. In addition to the native turn residues, the cyclic peptides include a linker unit between the N- and C-termini, selected by molecular modeling among various non-proteinogenic cyclic amino acids. NMR conformational studies showed that most of the cyclic peptides were able to adopt turn-like structures. Several of the analogues displayed significant inhibition of the BDNF-induced TrkB receptor phosphorylation, and hence could be useful templates for developing improved antagonists for this receptor.


Assuntos
Aminoácidos Cíclicos/química , Fator Neurotrófico Derivado do Encéfalo/química , Receptor trkB/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Desenho de Drogas , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Conformação Molecular , Peptídeos/química , Peptídeos Cíclicos/química , Fosforilação , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Temperatura Ambiente
10.
PLoS One ; 6(9): e24481, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21931728

RESUMO

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to ß-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.


Assuntos
Carbono-Nitrogênio Ligases/química , Fator de Iniciação 4G em Eucariotos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Solubilidade
11.
Org Biomol Chem ; 9(15): 5487-92, 2011 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-21670842

RESUMO

Trp-Trp pairs have emerged as a successful strategy for ß-hairpin stabilization. Using loop 3 of Vammin as a template, we experimentally demonstrate that the contribution of Trp-Trp pairs to ß-hairpin stability depends on ß-sheet periodicity, that is, they are stabilising at non-hydrogen-bonded sites, but not at hydrogen-bonded positions.


Assuntos
Hidrogênio/química , Triptofano/química , Sítios de Ligação , Ligações de Hidrogênio , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína
12.
Protein Eng Des Sel ; 24(1-2): 113-22, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21051322

RESUMO

The cell wall of Streptococcus pneumoniae and several other micro-organisms is decorated with a number of the so-called choline-binding proteins (CBPs) that recognise the choline residues in the bacterial surface by means of highly conserved, concatenated 20-aa sequences termed choline-binding repeats (CBRs), that are composed of a loop and a ß-hairpin structure. In this work, we have investigated the ability to fold in aqueous solution of a 14-aa peptide (LytA197₋210[wt]) and a single derivative of it, LytA197₋210[ND], corresponding to one of the six ß-hairpins of the LytA pneumococcal amidase. Intrinsic fluorescence and circular dichroism spectroscopical measurements showed that both peptides spontaneously acquire a non-random conformation which is also able to bind the natural ligand choline. Furthermore, nuclear magnetic resonance techniques allowed the calculation of the structure of the LytA197₋210[ND] peptide, which displayed a ß-hairpin conformation highly similar to that found within the full-length C-LytA module. These results provide a structural basis for the modular organisation of CBPs and suggest the use of CBRs as new templates for the design of stable ß-hairpins.


Assuntos
Amidoidrolases/química , Streptococcus pneumoniae/enzimologia , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Colina/metabolismo , Estabilidade Enzimática , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Streptococcus pneumoniae/química
13.
Biopolymers ; 94(6): 779-90, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20564027

RESUMO

Tryptophan plays important roles in protein stability and recognition despite its scarcity in proteins. Except as fluorescent groups, they have been used rarely in peptide design. Nevertheless, Trp residues were crucial for the stability of some designed minimal proteins. In 2000, Trp-Trp pairs were shown to contribute more than any other hydrophobic interaction to the stability of ß-hairpin peptides. Since then, Trp-Trp pairs have emerged as a paradigm for the design of stable ß-hairpins, such as the Trpzip peptides. Here, we analyze the nature of the stabilizing capacity of Trp-Trp pairs by reviewing the ß-hairpin peptides containing Trp-Trp pairs described up to now, the spectroscopic features and geometry of the Trp-Trp pairs, and their use as binding sites in ß-hairpin peptides. To complete the overview, we briefly go through the other relevant ß-hairpin stabilizing Trp-non-Trp interactions and illustrate the use of Trp in the design of short peptides adopting α-helical and mixed α/ß motifs. This review is of interest in the field of rational design of proteins, peptides, peptidomimetics, and biomaterials.


Assuntos
Peptídeos/química , Proteínas/química , Triptofano/química , Animais , Humanos , Peptídeos/genética , Peptidomiméticos/química , Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína , Proteínas/genética , Triptofano/genética
14.
J Med Chem ; 53(10): 4119-29, 2010 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-20411945

RESUMO

A series of gramicidin S (GS) analogues have been synthesized where the Phe (i + 1) and Pro (i + 2) residues of the beta-turn have been swapped while the respective chiralities (D-, L-) at each position are preserved, and Phe is replaced by surrogates with aromatic side chains of diverse size, orientation, and flexibility. Although most analogues preserve the beta-sheet structure, as assessed by NMR, their antibiotic activities turn out to be highly dependent on the bulkiness and spatial arrangement of the aromatic side chain. Significant increases in microbicidal potency against both Gram-positive and Gram-negative pathogens are observed for several analogues, resulting in improved therapeutic profiles. Data indicate that seemingly minor replacements at the GS beta-turn can have significant impact on antibiotic activity, highlighting this region as a hot spot for modulating GS plasticity and activity.


Assuntos
Antibacterianos/química , Gramicidina/análogos & derivados , Gramicidina/química , Fenilalanina/química , Animais , Antibacterianos/síntese química , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Gramicidina/síntese química , Gramicidina/farmacologia , Hemólise , Ligações de Hidrogênio , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Ovinos , Relação Estrutura-Atividade
15.
Nucleic Acids Res ; 38(15): 5226-41, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20410074

RESUMO

Direct targeting of critical DNA-binding elements of a repressor by its cognate antirepressor is an effective means to sequester the repressor and remove a transcription initiation block. Structural descriptions for this, though often proposed for bacterial and phage repressor-antirepressor systems, are unavailable. Here, we describe the structural and functional basis of how the Myxococcus xanthus CarS antirepressor recognizes and neutralizes its cognate repressors to turn on a photo-inducible promoter. CarA and CarH repress the carB operon in the dark. CarS, produced in the light, physically interacts with the MerR-type winged-helix DNA-binding domain of these repressors leading to activation of carB. The NMR structure of CarS1, a functional CarS variant, reveals a five-stranded, antiparallel beta-sheet fold resembling SH3 domains, protein-protein interaction modules prevalent in eukaryotes but rare in prokaryotes. NMR studies and analysis of site-directed mutants in vivo and in vitro unveil a solvent-exposed hydrophobic pocket lined by acidic residues in CarS, where the CarA DNA recognition helix docks with high affinity in an atypical ligand-recognition mode for SH3 domains. Our findings uncover an unprecedented use of the SH3 domain-like fold for protein-protein recognition whereby an antirepressor mimics operator DNA in sequestering the repressor DNA recognition helix to activate transcription.


Assuntos
Proteínas de Bactérias/química , Regiões Operadoras Genéticas , Proteínas Repressoras/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Fatores de Transcrição/genética , Domínios de Homologia de src
16.
Nat Struct Mol Biol ; 17(5): 617-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20400950

RESUMO

Trimethylation of Lys36 in histone H3 (H3K36me3) coordinates events associated with the elongation phase of transcription and is also emerging as an important epigenetic regulator of cell growth and differentiation. We have identified the PWWP domain of bromo and plant homeodomain (PHD) finger-containing protein 1 (BRPF1) as a H3K36me3 binding module and have determined the structure of this domain in complex with an H3K36me3-derived peptide.


Assuntos
Histonas/química , Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína
17.
Biomol NMR Assign ; 3(1): 9-12, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19636935

RESUMO

CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the (1)H, (13)C and (15)N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.


Assuntos
Proteínas de Bactérias/química , Espectroscopia de Ressonância Magnética/métodos , Myxococcus xanthus/química , Sequência de Aminoácidos , Isótopos de Carbono/química , Dados de Sequência Molecular , Isótopos de Nitrogênio/química , Estrutura Terciária de Proteína , Prótons
18.
Chembiochem ; 10(5): 902-10, 2009 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-19294654

RESUMO

Structural studies on model peptides have led to a good understanding of the rules behind the formation and stability of regular beta-hairpins. To test their applicability to the successful design of irregular beta-hairpins with long loops and/or beta-bulges at the strands, we mimicked loop 3 of vammin, a 4:6 beta-hairpin with a non-Gly beta-bulge. The most stabilising cross-strand pairs, disulfide bonds or/and TrpTrp pairs, were incorporated at non-hydrogen-bonded sites in peptides spanning the 69-80 region of vammin. According to NMR data, these modified peptides adopt beta-hairpin conformations as intended by design. The Trp-containing peptides reproduce even the unusual positive phi angle for the Gln residue, with the indole rings in the preferred edge-to-face orientation. For the first time the beta-hairpin-stabilising capacities of a disulfide bond and a TrpTrp pair are compared in the same model system. We found that the contribution to stability of the noncovalent indole-indole interaction is larger than that of the covalent disulfide bond, and that their combination gives rise to an even more stable beta-hairpin.


Assuntos
Dissulfetos/química , Peptídeos/química , Estrutura Secundária de Proteína , Triptofano/química , Fator A de Crescimento do Endotélio Vascular/química , Venenos de Víboras/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Alinhamento de Sequência
19.
J Med Chem ; 52(3): 664-74, 2009 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-19132829

RESUMO

Analogues of the cationic antimicrobial peptide gramicidin S (GS), cyclo(Val-Orn-Leu-D-Phe-Pro)2, with d-Phe residues replaced by different (restricted mobility, mostly) surrogates have been synthesized and used in SAR studies against several pathogenic bacteria. While all D-Phe substitutions are shown by NMR to preserve the overall beta-sheet conformation, they entail subtle structural alterations that lead to significant modifications in biological activity. In particular, the analogue incorporating D-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) shows a modest but significant increase in therapeutic index, mostly due to a sharp decrease in hemolytic effect. The fact that NMR data show a shortened distance between the D-Tic aromatic ring and the Orn delta-amino group may help explain the improved antibiotic profile of this analogue.


Assuntos
Gramicidina/análogos & derivados , Gramicidina/uso terapêutico , Fenilalanina/análogos & derivados , Acinetobacter baumannii/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Hemolíticos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Ressonância Magnética Nuclear Biomolecular , Fenilalanina/química , Estrutura Secundária de Proteína , Ovinos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Tetra-Hidroisoquinolinas/química
20.
Biochim Biophys Acta ; 1794(1): 110-7, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18955169

RESUMO

The first SH3 domain (SH3.1) of Nckalpha specifically recognizes the proline-rich region of CD3varepsilon, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckalpha SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel beta-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150-166 of CD3varepsilon binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3varepsilon reveals that Nckalpha SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position -2 relative to the WW sequence at the beginning of strand beta3 is crucial for PxxDY recognition. A 14-mer optimised Nckalpha SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckalpha SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckalpha SH3.1 binding relative to the wild type sequence.


Assuntos
Complexo CD3/química , Proteínas Oncogênicas/química , Peptídeos/química , Peptídeos/metabolismo , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Ligantes , Ativação Linfocitária , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Prolina/química , Prolina/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo
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