RESUMO
Arsenicals belong to the class of chemical warfare agents known as vesicants, which are highly reactive, toxic and cause robust inflammatory response. Cutaneous exposure to arsenicals causes a wide range of systemic organ damage, beginning with cutaneous injuries, and later manifest multi-organ damage and death. Thus, the development of suitable antidotes that can effectively block injury following exposure to these agents is of great importance. Bromodomain 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) family, plays crucial role in regulating transcription of inflammatory, proliferation and cell cycle genes. In this context, the development of potent small molecule inhibitors of BRD4 could serve as potential antidotes for arsenicals. Herein, we describe the synthesis and biological evaluation of a series of compounds.
Assuntos
Arsenicais , Anti-Inflamatórios/química , Antídotos/farmacologia , Arsenicais/farmacologia , Arsenicais/uso terapêutico , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3'-end-processing endonuclease CPSF-73, containing metallo-ß-lactamase and ß-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the ß-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.
Assuntos
Endonucleases/química , Dobramento de Proteína , Zinco/química , Animais , Domínio Catalítico , Proteínas de Ligação a DNA , Endonucleases/genética , Humanos , Células Sf9 , Spodoptera , Relação Estrutura-AtividadeRESUMO
Ca(2+) efflux by Ca(2+) cation antiporter (CaCA) proteins is important for maintenance of Ca(2+) homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na(+)/Ca(2+) exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca(2+) efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca(2+)/H(+) antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca(2+)-binding site. A hydrophobic "seal" closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca(2+)/H(+) exchange pathway. In the Ca(2+)-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na(+)-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca(2+)/H(+) exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.
Assuntos
Antiporters/química , Cálcio/metabolismo , Proteínas de Transporte de Cátions/química , Antiporters/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Transporte de Íons , Modelos MolecularesRESUMO
Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-ß-induced dysfunction in preclinical models of AD and also prevents amyloid-ß-independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain-containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain-containing proteins ameliorates amyloid-ß-induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-ß-induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.
Assuntos
Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Ensaios de Triagem em Larga EscalaRESUMO
CALX, the NCX homolog in Drosophila, involves in light-mediated Ca(2+) homeostasis in sensory neuronal cells. CALX exhibits a unique negative Ca(2+) regulatory property mediated by Ca2+ binding at its intracellular regulatory domain. Our structural studies of individual CBD1 or CBD2 domain reveal that CBD1 is the only Ca(2+) binding domain in CALX. Crystal structures of the entire Ca(2+) regulatory domain CBD12 from two alternative splicing isoforms, CALX1.1 and CALX1.2, demonstrate that CBD1 and CBD2 form an open V-shaped conformation with four Ca(2+) ions bound on the CBD domain interface. The structures together with Ca(2+) binding analyses strongly argue that the Ca(2+) inhibition of CALX is achieved by interdomain conformational change induced by Ca(2+) binding at CBD1. The conformational difference between the two isoforms also raises a hypothesis that alternative splicing residues adjust the interdomain orientation angle between CBD1 and CBD2 to modify the Ca(2+) regulatory property of the exchanger. These studies not only establish structural basis to understand the inhibitory Ca(2+) regulation and the alternative splicing modification of CALX, but also shed light on the general Ca(2+) regulatory mechanism of other mammalian NCX proteins.
Assuntos
Processamento Alternativo/fisiologia , Antiporters/química , Antiporters/metabolismo , Cálcio , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/metabolismo , Animais , Antiporters/genética , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Cátions , Proteínas de Drosophila/genética , Drosophila melanogaster , Transporte de Íons/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Trocador de Sódio e Cálcio/genética , Relação Estrutura-AtividadeRESUMO
Artemis is a 692 aa nuclease that is essential for opening hairpins during vertebrate V(D)J recombination. Artemis is also important in the DNA repair of double-strand breaks via the nonhomologous DNA end joining (NHEJ) pathway. Therefore, absence of Artemis has been shown to result not only in the blockage of lymphocyte development in vertebrates, but also sensitivity of organisms and cells to double-strand break-inducing events that arise in the course of normal metabolism. Nonhomologous DNA end joining (NHEJ) is the major pathway for the repair of double-strand DNA breaks in most vertebrate cells during most of the cell cycle, including in resting cells. Artemis is the primary nuclease for resection of damaged DNA at double-strand breaks. Artemis alone is inactive as an endonuclease, though it has 5'-exonuclease activity. The endonuclease activity requires physical interaction with DNA-PKcs and subsequent activation steps. Truncation of the C-terminal half of Artemis permits Artemis to be active, even without DNA-PKcs. Here we create a systematic set of deletions from the Artemis C-terminus to determine the minimal extent of C-terminal deletion for Artemis to function in a DNA-PKcs-independent manner. We discuss these data in the context of recent structural studies. The results will be useful in future studies to determine the full range of functions of the C-terminal region of Artemis in the regulation of its endonuclease activity.
Assuntos
Proteínas de Ligação a DNA , Proteínas Nucleares , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Endonucleases/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA/metabolismoRESUMO
Poly(A)-specific ribonuclease (PARN) is a mammalian 3'-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), approximately 10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.
Assuntos
Adenosina/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Aminoácidos/metabolismo , Biopolímeros/metabolismo , Domínio Catalítico , Humanos , Cinética , Nucleotídeos/metabolismo , Poli A/metabolismo , Estabilidade de RNA , Especificidade por SubstratoRESUMO
Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.
Assuntos
Antiporters/química , Antiporters/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Sódio/metabolismo , Animais , Antiporters/genética , Sítios de Ligação , Cristalografia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/fisiologiaRESUMO
Poly(A)-specific ribonuclease (PARN) is a homodimeric, processive, and cap-interacting 3' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. The crystal structure of a C-terminally truncated PARN in complex with m(7)GpppG reveals that, in one subunit, m(7)GpppG binds to a cavity formed by the RRM domain and the nuclease domain, whereas in the other subunit, it binds almost exclusively to the RRM domain. Importantly, our structural and competition data show that the cap-binding site overlaps with the active site in the nuclease domain. Mutational analysis demonstrates that residues involved in m(7)G recognition are crucial for cap-stimulated deadenylation activity, and those involved in both cap and poly(A) binding are important for catalysis. A modeled PARN, which shows that the RRM domain from one subunit and the R3H domain from the other subunit enclose the active site, provides a structural foundation for further studies to elucidate the mechanism of PARN-mediated deadenylation.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Animais , Sítios de Ligação , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Poli A/química , Poli A/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Capuzes de RNA/química , Capuzes de RNA/metabolismoRESUMO
The macrodomain of nsP3 (nsP3MD) is highly conserved among the alphaviruses and ADP-ribosylhydrolase activity of Chikungunya Virus (CHIKV) nsP3MD is critical for CHIKV viral replication and virulence. No small molecule drugs targeting CHIKV nsP3 have been identified to date. Here we report small fragments that bind to nsP3MD which were discovered by virtually screening a fragment library and X-ray crystallography. These identified fragments share a similar scaffold, 2-pyrimidone-4-carboxylic acid, and are specifically bound to the ADP-ribose binding site of nsP3MD. Among the fragments, 2-oxo-5,6-benzopyrimidine-4-carboxylic acid showed anti-CHIKV activity with an IC50 of 23 µM. Our fragment-based drug discovery approach provides valuable information to further develop a specific and potent nsP3 inhibitor of CHIKV viral replication based on the 2-pyrimidone-4-carboxylic acid scaffold. In silico studies suggest this pyrimidone scaffold could also bind to the macrodomains of other alphaviruses and coronaviruses and thus, have potential pan-antiviral activity.
Assuntos
Vírus Chikungunya/efeitos dos fármacos , Pirimidinonas/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Sítios de Ligação , Vírus Chikungunya/metabolismo , Desenho de Fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Proteínas não Estruturais Virais/metabolismoRESUMO
A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.
Assuntos
Antivirais/farmacologia , Derivados de Benzeno/química , Vírus Chikungunya/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/uso terapêutico , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Derivados de Benzeno/uso terapêutico , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Febre de Chikungunya/tratamento farmacológico , Di-Hidro-Orotato Desidrogenase , Modelos Animais de Doenças , Feminino , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Relação Estrutura-AtividadeRESUMO
Recruitment of the GRIP domain golgins to the trans-Golgi network is mediated by Arl1, a member of the ARF/Arl small GTPase family, through interaction between their GRIP domains and Arl1-GTP. The crystal structure of Arl1-GTP in complex with the GRIP domain of golgin-245 shows that Arl1-GTP interacts with the GRIP domain predominantly in a hydrophobic manner, with the switch II region conferring the main recognition surface. The involvement of the switch and interswitch regions in the interaction between Arl1-GTP and GRIP accounts for the specificity of GRIP domain for Arl1-GTP. Mutations that abolished the Arl1-mediated Golgi localization of GRIP domain golgins have been mapped on the interface between Arl1-GTP and GRIP. Notably, the GRIP domain forms a homodimer in which each subunit interacts separately with one Arl1-GTP. Mutations disrupting the GRIP domain dimerization also abrogated its Golgi targeting, suggesting that the dimeric form of GRIP domain is a functional unit.
Assuntos
Fatores de Ribosilação do ADP , Autoantígenos/química , Autoantígenos/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/genética , Sítios de Ligação , Cristalografia por Raios X , Dimerização , GTP Fosfo-Hidrolases/genética , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Proteínas de Membrana/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Rede trans-Golgi/metabolismoRESUMO
Oxygenated unsaturated fatty acids, known as oxylipins, are signaling molecules commonly used for cell-to-cell communication in eukaryotes. However, a role for oxylipins in mediating communication in prokaryotes has not previously been described. Bacteria mainly communicate via quorum sensing, which involves the production and detection of diverse small molecules termed autoinducers. Here we show that oleic acid-derived oxylipins produced by Pseudomonas aeruginosa function as autoinducers of a novel quorum sensing system. We found that this system controls the cell density-dependent expression of a gene subset independently of the quorum sensing systems thus far described in this bacterium. We identified a LysR-type transcriptional regulator as the primary receptor of the oxylipin signal. The discovery of this oxylipin-dependent quorum sensing system reveals that prokaryote-derived oxylipins also mediate cell-to-cell communication in bacteria.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Oxilipinas/metabolismo , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Carga Bacteriana , Fenômenos Fisiológicos Bacterianos/genética , Proteínas de Bactérias/metabolismo , Células Procarióticas/metabolismo , Células Procarióticas/fisiologia , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, ß-octyl glucoside (ßOG). X-ray crystallography revealed that a single ßOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-ßOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54-66. Nef dimer formation is stabilized in solution by the addition of ßOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.
Assuntos
Glucosídeos/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , HIV-1 , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/químicaRESUMO
The Na(+)/Ca(2+) exchanger CALX promotes Ca(2+) efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca(2+) homeostasis. CALX activity is negatively regulated by specific Ca(2+) interaction within its two intracellular Ca(2+) regulatory domains CBD1 and CBD2, yet how the Ca(2+) binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca(2+) regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.1 and 1.2, exhibiting distinct regulatory Ca(2+) dependency. The structures show an open V-shaped conformation with four Ca(2+) ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca(2+)-binding analysis support that the Ca(2+) inhibition of CALX is achieved by interdomain conformational changes induced by Ca(2+) binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca(2+) regulatory property of the exchangers.
Assuntos
Processamento Alternativo , Antiporters/química , Proteínas de Drosophila/química , Drosophila/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/química , Cromatografia em Gel , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Homologia de Sequência de Aminoácidos , Trocador de Sódio e Cálcio/químicaRESUMO
Na(+)/Ca(2+) exchangers (NCXs) promote the extrusion of intracellular Ca(2+) to terminate numerous Ca(2+)-mediated signaling processes. Ca(2+) interaction at two Ca(2+) binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca(2+) regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca(2+) interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca(2+) binding. The carboxylate residues that coordinate two Ca(2+) in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca(2+) regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca(2+) binding site to change Ca(2+) binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca(2+) regulatory behavior.
Assuntos
Processamento Alternativo , Cálcio/metabolismo , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Drosophila melanogaster , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Trocador de Sódio e Cálcio/genéticaRESUMO
Golgin-97, RanBP2alpha, Imh1p and p230/golgin-245 (GRIP) domain golgins are targeted to the Golgi membrane through their GRIP domains. By analyzing more than 30 mutants of golgin-97 and golgin-245 GRIP domains for their properties of dimerization, interaction with ARF like protein 1 (Arl1)-GTP and Golgi targeting, we found hierarchically organized three-tier interactions governing the Golgi targeting of GRIP domain golgins. GRIP domain self-dimerization is necessary for bivalent interaction with Arl1-GTP. Unexpectedly, however, these two interactions are not sufficient for Golgi targeting, as a third group of residues, including positive-charged arginine between alpha1 and alpha2 and hydrophobic residues C-terminal to the GRIP domain, turn out to be essential. Surface plasmon resonance analysis indicates that GRIP domain interacts directly with membrane lipid, partially through the third group of residues such as W744 of golgin-97. This third tier of interaction with the membrane could be mediated by non-specific hydrophobic and electrostatic forces.
Assuntos
Autoantígenos/metabolismo , Complexo de Golgi , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Autoantígenos/química , Autoantígenos/genética , Dimerização , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Lipídeos/química , Lipossomos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Rede trans-Golgi/metabolismoRESUMO
Rab7 regulates vesicle traffic from early to late endosomes, and from late endosomes to lysosomes. The crystal structure of Rab7-GTP in complex with the Rab7 binding domain of RILP reveals that Rab7 interacts with RILP specifically via two distinct areas, with the first one involving the switch and interswitch regions and the second one consisting of RabSF1 and RabSF4. Disruption of these interactions by mutations abrogates late endosomal/lysosomal targeting of Rab7 and RILP. The Rab7 binding domain of RILP forms a coiled-coil homodimer with two symmetric surfaces to interact with two separate Rab7-GTP molecules, forming a dyad configuration of Rab7-RILP(2)-Rab7. Mutations that disrupt RILP dimerization also abolish its interactions with Rab7-GTP and late endosomal/lysosomal targeting, suggesting that the dimeric form of RILP is a functional unit. Structural comparison suggests that the combined use of RabSF1 and RabSF4 with the switch regions may be a general mode of action for most Rab proteins in regulating membrane trafficking.
Assuntos
Proteínas de Transporte/química , Estrutura Quaternária de Proteína , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Dimerização , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Poly(A)-specific ribonuclease (PARN) is a processive, poly(A)-specific 3' exoribonuclease. The crystal structure of C-terminal truncated human PARN determined in two states (free and RNA-bound forms) reveals that PARNn is folded into two domains, an R3H domain and a nuclease domain similar to those of Pop2p and epsilon186. The high similarity of the active site structures of PARNn and epsilon186 suggests that they may have a similar catalytic mechanism. PARNn forms a tight homodimer, with the R3H domain of one subunit partially enclosing the active site of the other subunit and poly(A) bound in a deep cavity of its nuclease domain in a sequence-nonspecific manner. The R3H domain and, possibly, the cap-binding domain are involved in poly(A) binding but these domains alone do not appear to contribute to poly(A) specificity. Mutations disrupting dimerization abolish both the enzymatic and RNA-binding activities, suggesting that the PARN dimer is a structural and functional unit. The cap-binding domain may act in concert with the R3H domain to amplify the processivity of PARN.