RESUMO
A series of fourteen 2-aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones was prepared at room temperature by T3P-mediated cyclization of N-phenyl-C-aryl imines with thionicotinic acid, two difficult substrates. The reactions were operationally simple, did not require specialized equipment or anhydrous solvents, could be performed as either two or three component reactions, and gave moderate-good yields as high as 63%. This provides ready access to N-phenyl compounds in this family, which have been generally difficult to prepare. As part of the study, the first crystal structure of neutral thionicotinic acid is also reported, and showed the molecule to be in the form of the thione tautomer. Additionally, the synthesized compounds were tested against T. brucei, the causative agent of Human African Sleeping Sickness. Screening at 50 µM concentration showed that five of the compounds strongly inhibited growth and killed parasites.
Assuntos
Tiazinas , Trypanosoma brucei brucei/efeitos dos fármacos , Anidridos/química , Animais , Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Organofosfonatos/química , Tiazinas/síntese química , Tiazinas/farmacologiaRESUMO
3-Phenyl-2-(thio-phen-3-yl)-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-one (C17H12N2OS2, 1) and 2-(1H-indol-3-yl)-3-phenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-one 0.438-hydrate (C21H15N3OS·0.438H2O, 2) crystallize in space groups P21/n and C2/c, respectively. The asymmetric unit in each case is comprised of two parent mol-ecules, albeit of mixed chirality in the case of 1 and of similar chirality in 2 with the enanti-omers occupying the neighboring asymmetric units. Structure 2 also has water mol-ecules (partial occupancies) that form continuous channels along the b -axis direction. The thia-zine rings in both structures exhibit an envelope conformation. Inter-molecular inter-actions in 1 are defined only by C-Hâ¯O and C-Hâ¯N hydrogen bonds between crystallographically independent mol-ecules. In 2, hydrogen bonds of the type N-Hâ¯O between independent mol-ecules and C-Hâ¯N(π) type, and π-π stacking inter-actions between the pyridine rings of symmetry-related mol-ecules are observed.
RESUMO
The title sulfones, 2,3-diphenyl-2,3-di-hydro-4H-1,3-benzo-thia-zine-1,1,4-trione, C20H15NO3S, and 2,3-diphenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zine-1,1,4-trione, C19H14N2O3S, crystallize in space group P21/n with two mol-ecules in each of the asymmetric units and have almost identical unit cells and extended structures. In both structures, the thia-zine rings exhibit a screw-boat pucker. The inter-molecular inter-actions observed are C-Hâ¯O-type hydrogen bonds and parallel partial π-π stacking between the fused aromatic rings (benzo- or pyrido-) of the core of the mol-ecules within each asymmetric unit, and also connecting to mol-ecules with translational periodicity in the a-axis direction in what can be described as columns (two per asymmetric unit) of stacked mol-ecules with alternating chirality. The pendant phenyl groups of both mol-ecules do not participate in aromatic ring inter-actions.
RESUMO
The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP(3)R) exhibit distinct IP(3) sensitivities and cooperativities in calcium (Ca(2+)) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP(3)R. We determined the 1.9 Å crystal structure of the suppressor domain from type 3 IP(3)R (IP(3)R3(SUP), amino acids 1-224) and revealed structural features contributing to isoform-specific functionality of IP(3)R by comparing it with our previously determined structure of the type 1 suppressor domain (IP(3)R1(SUP)). The molecular surface known to associate with the ligand binding domain (amino acids 224-604) showed marked differences between IP(3)R3(SUP) and IP(3)R1(SUP). Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP(3)R1 and Glu-19, Trp-168, and Ser-218 in IP(3)R3) within the N-terminal suppressor domains of IP(3)R1(SUP) and IP(3)R3(SUP) interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081-36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP(3)R1 and Trp-168 in IP(3)R3) plays a critical role in the coupling between ligand binding and channel gating.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cristalografia por Raios X , Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Triptofano/química , Triptofano/genética , Triptofano/metabolismo , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMO
NMR spectroscopy has become one of the preferred analytical techniques for metabolomics studies due to its inherent nondestructive nature, ability to identify and quantify metabolites simultaneously in a complex mixture, minimal sample preparation requirement, and high degree of experimental reproducibility. NMR-based metabolomics studies involve the measurement and multivariate statistical analysis of metabolites present in biological samples such as biofluids, stool/feces, intestinal content, tissue, and cell extracts by high-resolution NMR spectroscopy-the goal then is to identify and quantify metabolites and evaluate changes of metabolite concentrations in response to some perturbation. Here we describe methodologies for NMR sample preparation of biofluids (serum, saliva, and urine) and stool/feces, intestinal content, and tissues for NMR experiments including extraction of polar metabolites and application of NMR in metabolomics studies. One dimensional (1D) 1H NMR experiments with different variations such as pre-saturation, relaxation-edited, and diffusion-edited are routinely acquired for profiling and metabolite identification and quantification. 2D homonuclear 1H-1H TOCSY and COSY, 2D J-resolved, and heteronuclear 1H-13C HSQC and HMBC are also performed to assist with metabolite identification and quantification. The NMR data are then subjected to targeted and/or untargeted multivariate statistical analysis for biomarker discovery, clinical diagnosis, toxicological studies, molecular phenotyping, and functional genomics.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Manejo de Espécimes/métodos , Animais , Líquidos Corporais/química , Fezes/química , Humanos , Análise Multivariada , Reprodutibilidade dos Testes , Saliva/química , Soro/química , Urina/químicaRESUMO
Precision antimicrobials aim to kill pathogens without damaging commensal bacteria in the host, and thereby cure disease without antibiotic-associated dysbiosis. Here we report the de novo design of a synthetic host defence peptide that targets a specific pathogen by mimicking key molecular features of the pathogen's channel-forming membrane proteins. By exploiting physical and structural vulnerabilities within the pathogen's cellular envelope, we designed a peptide sequence that undergoes instructed tryptophan-zippered assembly within the mycolic acid-rich outer membrane of Mycobacterium tuberculosis to specifically kill the pathogen without collateral toxicity towards lung commensal bacteria or host tissue. These mycomembrane-templated assemblies elicit rapid mycobactericidal activity and enhance the potency of antibiotics by improving their otherwise poor diffusion across the rigid M. tuberculosis envelope with respect to agents that exploit transmembrane protein channels for antimycobacterial activity. This biomimetic strategy may aid the design of other narrow-spectrum antimicrobial peptides.
Assuntos
Antibacterianos/farmacologia , Proteínas de Membrana/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Humanos , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Mimetismo Molecular , Peptídeos/genéticaRESUMO
Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.
Assuntos
Receptores de Inositol 1,4,5-Trifosfato/química , Animais , Cálcio/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ligantes , Camundongos , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica , Estrutura Secundária de Proteína , Análise Espectral , Difração de Raios XRESUMO
MazF and MazE constitute a so-called addiction module that is critical for bacterial growth arrest and eventual cell death in response to stress. The MazF toxin was recently shown to possess mRNA interferase (MIase) activity, and acts as a protein synthesis inhibitor by cleaving cellular mRNA. As a cognate regulator, the short-lived antitoxin, MazE, inhibits MazF MIase activity and hence maintains the delicate homeostasis between these two components. In the present study, we have shown that the MazF homodimer contains two symmetric binding sites, each of which is capable of interacting with a MazE C-terminal peptide, MazEp(54-77). The slow exchange phenomenon between free and peptide-bound MazF on the NMR timescale indicates relatively high affinities for MazEp(54-77) at both sites (Kd,K'd < 10(-7) M). However, the observed sequential binding behavior suggests a negative cooperativity between the two sites (Kd < K'd). A 13 base single-stranded DNA, employed as an uncleavable RNA substrate analog, can also bind to both sites on the MazF homodimer with moderate affinity (Kd approximately 10(-5) -10(-6) M). Chemical shift perturbation data deduced from NMR experiments indicates that the two binding sites for the MazEp peptide coincided with those for the single-stranded DNA competitive inhibitor. These dual substrate-binding sites are located on the concave interface of the MazF homodimer, consisting of a highly basic region underneath the S1-S2 loop and two hydrophobic regions containing the H1 helix of one subunit and the S3-S4 loop of the opposing subunit. We show that the MazF homodimer is a bidentate endoribonuclease equipped with two identical binding sites for mRNA processing and that a single MazE molecule occupying one of the binding sites can affect the conformation of both sites, hence efficiently hindering the activity of MazF.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Estrutura Quaternária de Proteína , Sequência de Aminoácidos , Sítios de Ligação , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Endorribonucleases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Histidina/química , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.
Assuntos
Proteínas de Bactérias/genética , Temperatura Baixa , Escherichia coli/genética , Vetores Genéticos/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/metabolismo , Humanos , Marcação por Isótopo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMO
It was recently discovered that the NRAS isoform 5 (20 amino acids) is expressed in melanoma and results in a more aggressive cell phenotype. This novel isoform is responsible for increased phosphorylation of downstream targets such as AKT, MEK, and ERK as well as increased cellular proliferation. This structure report describes the NMR solution structure of NRAS isoform 5 to be used as a starting point to understand its biophysical interactions. The isoform is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Trifluoretanol/química , Dicroísmo Circular , Humanos , Melanoma/genética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Conformação Proteica em alfa-Hélice , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Neoplasias Cutâneas/genéticaRESUMO
General transcription factor TFIID, consisting of TATA-binding protein (TBP) and TBP-associated factors (TAFs), plays a central role in both positive and negative regulation of transcription. The TAF N-terminal domain (TAND) of TAF1 has been shown to interact with TBP and to modulate the interaction of TBP with the TATA box, which is required for transcriptional initiation and activation of TATA-promoter operated genes. We have previously demonstrated that the Drosophila TAND region of TAF1 (residues 11-77) undergoes an induced folding from a largely unstructured state to a globular structure that occupies the DNA-binding surface of TBP thereby inhibiting the DNA-binding activity of TBP. In Saccharomyces cerevisiae, the TAND region of TAF1 displays marked differences in the primary structure relative to Drosophila TAF1 (11% identity) yet possesses transcriptional activity both in vivo and in vitro. Here we present structural and functional studies of yeast TAND1 and TAND2 regions (residues 10-37, and 46-71, respectively). Our NMR data show that, in yeast, TAND1 contains two alpha-helices (residues 16-23, 30-36) and TAND2 forms a mini beta-sheet structure (residues 53-56, 61-64). These TAND1 and TAND2 structured regions interact with the concave and convex sides of the saddle-like structure of TBP, respectively. Present NMR, mutagenesis and genetic data together elucidate that the minimal region (TAND1 core) required for GAL4-dependent transcriptional activation corresponds to the first helix region of TAND1, while the functional core region of TAND2, involved in direct interaction with TBP convex alpha-helix 2, overlaps with the mini beta-sheet region.
Assuntos
Saccharomyces cerevisiae/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box/química , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Activation of glutamate decarboxylase (GAD) by calcium-bound calmodulin (CaM) is required for normal plant growth through regulation of gamma-aminobutyrate and glutamate metabolism. The interaction of CaM with the C-terminal domain of GAD is believed to induce dimerization of the enzyme, an event implicated for Ca(2+)-dependent enzyme activation. Here, we present the solution structure of CaM in complex with a dimer of peptides derived from the C-terminus of Petunia hybrida GAD. The 23 kDa ternary complex is pseudo-symmetrical with each domain of CaM bound to one of the two antiparallel GAD peptides, which form an X-shape with an interhelical angle of 60 degrees. To accommodate the dimeric helical GAD target, the two domains of CaM adopt an orientation markedly different from that seen in other CaM-target complexes. Although the dimeric GAD domain is much larger than previously studied CaM-binding peptides, the two CaM domains appear closer together and make a number of interdomain contacts not observed in earlier complexes. The present structure of a single CaM molecule interacting with two target peptides provides new evidence for the conformational flexibility of CaM as well as a structural basis for the ability of CaM to activate two enzyme molecules simultaneously.
Assuntos
Calmodulina/metabolismo , Glutamato Descarboxilase/química , Glutamato Descarboxilase/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Calmodulina/química , Dimerização , Imageamento Tridimensional , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Petunia/enzimologia , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II.
Assuntos
Fator de Transcrição TFIIB/química , Zinco/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cátions Bivalentes/metabolismo , Endopeptidases/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Fator de Transcrição TFIIB/metabolismoRESUMO
General transcription factor TFIID is comprised of TATA-binding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10-37) and TAND2 (residues 46-71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6-71) was hindered by its poor solubility and stability in solution. Here we report a molecular engineering approach where the N terminus of TBP is fused to the C terminus of TAND12 via linkers of various lengths containing (GGGS)(n) sequence, (n = 1, 2, 3). The length of the linker within the TAND12-TBP fusion has a significant effect on solubility and stability (SAS). The construct with (GGGS)(3) linker produces the best quality single-quantum-coherence (HSQC) NMR spectrum with markedly improved SAS. In parallel to these observations, the TAND12-TBP fusion exhibits marked reduction of TBP function in binding to TAF1 as well as temperature sensitivity in in vivo yeast cell growth. Remarkably, the temperature sensitivity was proportional to the length of the linker in the fusions: the construct with (GGGS)(3) linker did not grow at 20 degrees C, while those with (GGGS)(1) and (GGGS)(2) linkers did. These results together indicate that the native interaction between TBP and TAND12 is well maintained in the TAND12-(GGGS)(3)-TBP fusion and that this fusion approach provides an excellent model system to investigate the structural detail of the TBP-TAF1 interaction.
Assuntos
Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/antagonistas & inibidores , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genética , Termodinâmica , Fator de Transcrição TFIID/antagonistas & inibidoresRESUMO
The utility of a three-residue Cu2+ binding motif (ATCUN domain) for studying intermolecular interactions is demonstrated. By comparing a set of 1H-15N correlation spectra recorded on complexes of calmodulin (CaM) and peptides with the ATCUN tag in the presence and absence of Cu2+ the two possible canonical binding orientations of the peptide can be rapidly distinguished. The methodology is confirmed with studies of complexes of CaM and peptides from myosin light chain kinase and CaM kinase kinase, for which high-resolution structures are available, and then applied to a complex with CaM kinase I for which structural data has not been obtained. The orientation of the CaM kinase I and myosin light chain kinase peptides are shown to be identical. In the case of a complex of CaM with a peptide for which structural information is not available, the present methodology, in combination with 1H-15N residual dipolar couplings measured on CaM, and the database of existing CaM-peptide structures, allows a homology model to be built rapidly and with confidence.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Calmodulina/química , Quinase de Cadeia Leve de Miosina/química , Sequência de Aminoácidos , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) mediates muscle relaxation by pumping Ca(2+) from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , ATPases Transportadoras de Cálcio/química , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Homologia de Sequência de AminoácidosRESUMO
Calmodulin-regulated serine/threonine kinases (CaM kinases) play crucial roles in Ca2+-dependent signaling transduction pathways in eukaryotes. Despite having a similar overall molecular architecture of catalytic and regulatory domains, CaM kinases employ different binding modes for Ca2+/CaM recruitment which is required for their activation. Here we present a residual dipolar coupling (RDC)-based NMR approach to characterizing the molecular recognition of CaM with five different CaM kinases. Our analyses indicate that CaM kinase I and likely IV use the same CaM binding mode as myosin light chain kinase (1-14 motif), distinct from those of CaM kinase II (1-10 motif) and CaM kinase kinase (1-16- motif). This NMR approach provides an efficient experimental guide for homology modeling and structural characterization of CaM-target complexes.
Assuntos
Calmodulina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Quinases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/química , Isótopos de Nitrogênio , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Prótons , Soluções , Xenopus laevisRESUMO
The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian, and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylated forms of the MUC1 tandem repeat. In this report, (15)N- and (13)C-labeled forms of a recombinant MUC1 construct containing five tandem repeats were used as substrates to define the order and kinetics of addition of N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). Heteronuclear NMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15) and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kinetics and order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1 were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylated by ppGalNAc-T1 with an initial rate of 25 microM/min, followed by Thr6 (8.6 microM/min). The enzyme also modified Ser5 at a slower rate (1.7 microM/min), an event that started only after the glycosylation of Thr14 and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified by ppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This study demonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominant substrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. More importantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relative simplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylation pathways and the rational design of tumor-associated MUC1 antigens.
Assuntos
Mucina-1/química , N-Acetilgalactosaminiltransferases/química , Sequências Repetitivas de Aminoácidos , Acetilgalactosamina/química , Sequência de Aminoácidos , Sequência de Carboidratos , Domínio Catalítico/genética , Vetores Genéticos , Glicosilação , Histidina/genética , Humanos , Dados de Sequência Molecular , Mucina-1/biossíntese , Mucina-1/genética , N-Acetilgalactosaminiltransferases/genética , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Sequências Repetitivas de Aminoácidos/genética , Solubilidade , Especificidade por Substrato/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-A crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.