RESUMO
General control nonderepressible 5 (GCN5, also known as Kat2a) and p300/CBP-associated factor (PCAF, also known as Kat2b) are two homologous acetyltransferases. Both proteins share similar domain architecture consisting of a PCAF N-terminal (PCAF_N) domain, acetyltransferase domain, and a bromodomain. PCAF also acts as a ubiquitin E3 ligase whose activity is attributable to the PCAF_N domain, but its structural aspects are largely unknown. Here, we demonstrated that GCN5 exhibited ubiquitination activity in a similar manner to PCAF and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which was not predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides new insight into not only the functional significance of GCN5 but also into ubiquitin biology.
Assuntos
Ubiquitina-Proteína Ligases/química , Fatores de Transcrição de p300-CBP/química , Animais , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.
Assuntos
Fator Gênico 3 Estimulado por Interferon, Subunidade gama/química , Vírus do Sarampo/metabolismo , Fosfoproteínas/química , Fator de Transcrição STAT2/química , Proteínas Virais/química , Sítios de Ligação , Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Janus Quinases/metabolismo , Vírus do Sarampo/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Fator de Transcrição STAT1/química , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/metabolismo , Dedos de ZincoRESUMO
The complex between Trm7 and Trm734 (Trm7-Trm734) from Saccharomyces cerevisiae catalyzes 2'-O-methylation at position 34 in tRNA. We report biochemical and structural studies of the Trm7-Trm734 complex. Purified recombinant Trm7-Trm734 preferentially methylates tRNAPhe transcript variants possessing two of three factors (Cm32, m1G37 and pyrimidine34). Therefore, tRNAPhe, tRNATrp and tRNALeu are specifically methylated by Trm7-Trm734. We have solved the crystal structures of the apo and S-adenosyl-L-methionine bound forms of Trm7-Trm734. Small angle X-ray scattering reveals that Trm7-Trm734 exists as a hetero-dimer in solution. Trm7 possesses a Rossmann-fold catalytic domain, while Trm734 consists of three WD40 ß-propeller domains (termed BPA, BPB and BPC). BPA and BPC form a unique V-shaped cleft, which docks to Trm7. The C-terminal region of Trm7 is required for binding to Trm734. The D-arm of substrate tRNA is required for methylation by Trm7-Trm734. If the D-arm in tRNAPhe is docked onto the positively charged area of BPB in Trm734, the anticodon-loop is located near the catalytic pocket of Trm7. This model suggests that Trm734 is required for correct positioning of tRNA for methylation. Additionally, a point-mutation in Trm7, which is observed in FTSJ1 (human Trm7 ortholog) of nosyndromic X-linked intellectual disability patients, decreases the methylation activity.
Assuntos
RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Transporte Vesicular/química , tRNA Metiltransferases/química , Domínio Catalítico , Ligação de Hidrogênio , Metilação , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Pirimidinas/metabolismo , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Proteínas de Transporte Vesicular/metabolismo , tRNA Metiltransferases/metabolismoRESUMO
In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V(1) portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A(3)B(3) complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V(1)-ATPase from the A(3)B(3) and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A(3)B(3) complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V(1)-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V(1)-ATPase.
Assuntos
Enterococcus/enzimologia , Modelos Moleculares , ATPases Vacuolares Próton-Translocadoras/química , Sítios de Ligação , Cristalização , Enterococcus/genética , Mutação , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Rotação , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Small GTPases are molecular switches that have critical biological roles and are controlled by GTPase-activating proteins and guanine nucleotide exchange factors (GEFs). The smg GDP dissociation stimulator (SmgGDS) protein functions as a GEF for the RhoA and RhoC small GTPases. SmgGDS has various regulatory roles, including small GTPase trafficking and localization and as a molecular chaperone, and interacts with many small GTPases possessing polybasic regions. Two SmgGDS splice variants, SmgGDS-558 and SmgGDS-607, differ in GEF activity and binding affinity for RhoA depending on the lipidation state, but the reasons for these differences are unclear. Here we determined the crystal structure of SmgGDS-558, revealing a fold containing tandem copies of armadillo repeats not present in other GEFs. We also observed that SmgGDS harbors distinct positively and negatively charged regions, both of which play critical roles in binding to RhoA and GEF activity. This is the first report demonstrating a relationship between the molecular function and atomic structure of SmgGDS. Our findings indicate that the two SmgGDS isoforms differ in GTPase binding and GEF activity, depending on the lipidation state, thus providing useful information about the cellular functions of SmgGDS in cells.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Modelos Moleculares , Prenilação de Proteína , Proteína rhoA de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Farnesiltranstransferase/genética , Farnesiltranstransferase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Solubilidade , Ressonância de Plasmônio de Superfície , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
In vitro transcription systems have been utilized to elucidate detailed mechanisms of transcription. Purified RNA polymerase II (pol II) and general transcription factors (GTFs) are required for the in vitro reconstitution of eukaryotic transcription systems. Among GTFs, TFIID and TFIIA play critical roles in the early stage of transcription initiation; TFIID first binds to the DNA in transcription initiation and TFIIA regulates TFIID's DNA binding activity. Despite the important roles of TFIIA, the time-consuming steps required to purify it, such as denaturing and refolding, have hampered the preparation of in vitro transcription systems. Here, we report an improved method for soluble expression and rapid purification of yeast TFIIA. The subunits of TFIIA, TOA1 and TOA2, were bacterially expressed as fusion proteins in soluble form, then processed by the PreScission protease and co-purified. TFIIA's heterodimer formation was confirmed by size exclusion chromatography-multiangle light scattering (SEC-MALS). The hydrodynamic radius (Rh) and radius of gyration (Rg) were measured by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), respectively. The Rg/Rh value implied that the intrinsically disordered region of TOA1 might not have an extended structure in solution. Our improved method provides highly purified TFIIA of sufficient quality for biochemical, biophysical, and structural analyses of eukaryotic transcription systems.
Assuntos
Escherichia coli/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIA , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade , Fator de Transcrição TFIIA/biossíntese , Fator de Transcrição TFIIA/química , Fator de Transcrição TFIIA/genéticaRESUMO
The mammalian peripheral stalk subunits of the vacuolar-type H(+)-ATPases (V-ATPases) possess several isoforms (C1, C2, E1, E2, G1, G2, G3, a1, a2, a3, and a4), which may play significant role in regulating ATPase assembly and disassembly in different tissues. To better understand the structure and function of V-ATPase, we expressed and purified several isoforms of the human V-ATPase peripheral stalk: E1G1, E1G2, E1G3, E2G1, E2G2, E2G3, C1, C2, H, a1NT, and a2NT. Here, we investigated and characterized the isoforms of the peripheral stalk region of human V-ATPase with respect to their affinity and kinetics in different combination. We found that different isoforms interacted in a similar manner with the isoforms of other subunits. The differences in binding affinities among isoforms were minor from our in vitro studies. However, such minor differences from the binding interaction among isoforms might provide valuable information for the future structural-functional studies of this holoenzyme.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sistema Livre de Células/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long left-handed coiled coil with a unique short ß-hairpin region that is effective in stimulating the ATPase activity of V(1)-ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A(3)B(3) complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.
Assuntos
Enterococcus/enzimologia , Células Procarióticas/enzimologia , Subunidades Proteicas/química , ATPases Vacuolares Próton-Translocadoras/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Eletricidade Estática , Homologia Estrutural de Proteína , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The selective inhibition of an aminopeptidase from Aeromonas proteolytica (AAP), a dinuclear Zn(2+) hydrolase, by 8-quinolinol (8-hydroxyquinoline, 8-HQ) derivatives is reported. We previously reported on the preparation of 8-HQ-pendant cyclens as Zn(2+) fluorophores (cyclen is 1,4,7,10-tetraazacyclododecane), in which the nitrogen and phenolate of the 8-HQ units (as well as the four nitrogens of cyclen) bind to Zn(2+) in a bidentate manner to form very stable Zn(2+) complexes at neutral pH (K (d) = 8-50 fM at pH 7.4). On the basis of this finding, it was hypothesized that 8-HQ derivatives have the potential to function as specific inhibitors of Zn(2+) enzymes, especially dinuclear Zn(2+) hydrolases. Assays of 8-HQ derivatives as inhibitors were performed against commercially available dinuclear Zn(2+) enzymes such as AAP and alkaline phosphatase. 8-HQ and the 5-substituted 8-HQ derivatives were found to be competitive inhibitors of AAP with inhibition constants of 0.16-29 µM at pH 8.0. The nitrogen at the 1-position and the hydroxide at the 8-position of 8-HQ were found to be essential for the inhibition of AAP. Fluorescence titrations of these drugs with AAP and an X-ray crystal structure analysis of an AAP-8-HQ complex (1.3-Å resolution) confirmed that 8-HQ binds to AAP in the "Pyr-out" mode, in which the hydroxide anion of 8-HQ bridges two Zn(2+) ions (Zn1 and Zn2) in the active site of AAP and the nitrogen atom of 8-HQ coordinates to Zn1 (Protein Data Bank code 3VH9).
Assuntos
Aeromonas/enzimologia , Aminopeptidases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Compostos Organometálicos/farmacologia , Oxiquinolina/farmacologia , Aminopeptidases/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Oxiquinolina/síntese química , Oxiquinolina/química , Teoria Quântica , Estereoisomerismo , Relação Estrutura-Atividade , Zinco/químicaRESUMO
AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/enzimologia , Humanos , Síndrome Metabólica/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Obesidade/enzimologia , Estrutura Terciária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Alinhamento de SequênciaRESUMO
Biacore is widely used for studies on protein-protein interaction in which regeneration is one of the most important steps. Here we introduce the anionic detergent sodium lauroyl sarcosinate (sarkosyl), which works satisfactorily as a regeneration reagent. After regeneration by the mild detergent, the subsequent binding experiment was reproducible without any degradation of the ligand. This regeneration condition can be employed for diverse combinations of ligand-analyte binding interactions and optimized as required.
Assuntos
Detergentes/farmacologia , Sarcosina/análogos & derivados , ATPases Vacuolares Próton-Translocadoras/análise , ATPases Vacuolares Próton-Translocadoras/metabolismo , Detergentes/química , Humanos , Ligantes , Ligação Proteica , Sarcosina/química , Sarcosina/farmacologia , ATPases Vacuolares Próton-Translocadoras/químicaRESUMO
The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.
Assuntos
Proteínas Recombinantes/química , ATPases Vacuolares Próton-Translocadoras/química , Sequência de Aminoácidos , Sistema Livre de Células , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Dados de Sequência Molecular , Isoformas de Proteínas , Multimerização Proteica , Subunidades Proteicas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de Proteína , ATPases Vacuolares Próton-Translocadoras/biossíntese , ATPases Vacuolares Próton-Translocadoras/isolamento & purificaçãoRESUMO
Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V(1); NtpA(3)-B(3)-D-G) and an integral membrane domain (V(0); NtpI-K(10)) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V(1) portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 microM for ADP or 3.1 microM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA(3)-B(3) heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA(3)-B(3) forming V(1) (NtpA(3)-B(3)-D-G) complex independent of nucleotides. The V(1) formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V(1) complex was as high as that of native V(1)-ATPase purified from the V(0)V(1) complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V(1) complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V(1)-ATPase complex.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Nucleotídeos/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Escherichia coli/química , Escherichia coli/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificaçãoRESUMO
Microtubules (MTs) are dynamic structures that are fundamental for cell morphogenesis and motility. MT-associated motors work efficiently to perform their functions. Unlike other motile kinesins, KIF2 catalytically depolymerizes MTs from the peeled protofilament end during ATP hydrolysis. However, the detailed mechanism by which KIF2 drives processive MT depolymerization remains unknown. To elucidate the catalytic mechanism, the transitional KIF2-tubulin complex during MT depolymerization was analyzed through multiple methods, including atomic force microscopy, size-exclusion chromatography, multi-angle light scattering, small-angle X-ray scattering, analytical ultracentrifugation, and mass spectrometry. The analyses outlined the conformation in which one KIF2core domain binds tightly to two tubulin dimers in the middle pre-hydrolysis state during ATP hydrolysis, a process critical for catalytic MT depolymerization. The X-ray crystallographic structure of the KIF2core domain displays the activated conformation that sustains the large KIF2-tubulin 1:2 complex.
Assuntos
Biocatálise , Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Polimerização , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Hidrólise , Cinesinas/genética , Mutação com Perda de Função , Modelos Moleculares , Peso Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca(2+)-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca(2+) at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.
Assuntos
Hidrolases/química , Arginina/química , Sítios de Ligação/fisiologia , Cristalografia por Raios X/métodos , Humanos , Isoenzimas/química , Proteína-Arginina Desiminase do Tipo 1 , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Raios XRESUMO
The reaction center-light-harvesting 1 (RC-LH1) core complex is the photosynthetic apparatus in the membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. The RC is surrounded by an LH1 complex that is constituted of oligomers of three types of apoproteins (alpha, beta and gamma chains) with associated bacteriochlorophyll bs and carotenoid. It has been crystallized by the sitting-drop vapour-diffusion method. A promising crystal diffracted to beyond 8.0 A resolution. It belonged to space group P1, with unit-cell parameters a = 141.4, b = 136.9, c = 185.3 A, alpha = 104.6, beta = 94.0, gamma = 110.7 degrees. A Patterson function calculated using data between 15.0 and 8.0 A resolution suggested that the LH1 complex is distributed with quasi-16-fold rotational symmetry around the RC.
Assuntos
Complexos de Proteínas Captadores de Luz/metabolismo , Rodopseudomonas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Cristalização , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Conformação Proteica , Difração de Raios XRESUMO
The growth of suitably sized protein crystals is essential for protein structure determination by X-ray crystallography. In general, crystals are grown using a trial-and-error method. However, these methods have been modified with the advent of microlitre dispensing-robot technology and of protocols that rapidly screen for crystal nucleation conditions. The use of one such automatic dispenser for mixing protein drops (1.3-2.0 microl in volume) of known concentration and pH with precipitating solutions (ejecting 2.0 microl droplets) containing salt is described here. The results of the experiments are relevant to a crystallization approach based on a two-step procedure: screening for the crystal nucleation step employing robotics followed by optimization of the crystallization conditions using incomplete factorial experimental design. Large crystals have successfully been obtained using quantities as small as 3.52 mg protein.
Assuntos
Basidiomycota/enzimologia , Corantes/metabolismo , Peroxidase/metabolismo , Precipitação Química , Cristalização , Glicosilação , Concentração Osmolar , Difração de Raios XRESUMO
Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (â¼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.
Assuntos
ATPases Vacuolares Próton-Translocadoras/metabolismo , Biofísica , Cromatografia em Gel , Humanos , Eletroforese em Gel de Poliacrilamida Nativa , Ligação Proteica , Ressonância de Plasmônio de Superfície , ATPases Vacuolares Próton-Translocadoras/químicaRESUMO
Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcushirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex.
Assuntos
Enterococcus/enzimologia , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Modelos Moleculares , Proteínas Mutantes/metabolismo , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV(476-7)AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity.