RESUMO
Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8â Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by â¼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by â¼60-fold in the absence of Na+ and by â¼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.
Assuntos
Cálcio/metabolismo , Fator VIIa , Magnésio/metabolismo , Rubídio/metabolismo , Sítios de Ligação , Fator VIIa/química , Fator VIIa/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Homology threading is a powerful technology for generating structural models based on homologous structures. Here we use threading to generate four complex RNA polymerase models. The models appear to be as useful as x-ray crystal structures or cryo-electron microscopy structures to support research projects.
Assuntos
Biologia Computacional/métodos , RNA Polimerases Dirigidas por DNA/química , Modelos Moleculares , Conformação Proteica , Animais , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/ultraestruturaRESUMO
Crystal structures of factor (F) VIIa/soluble tissue factor (TF), obtained under high Mg(2+) (50mM Mg(2+)/5mM Ca(2+)), have three of seven Ca(2+) sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg(2+) at positions 1, 4, and 7. We now report structures under low Mg(2+) (2.5mM Mg(2+)/5mM Ca(2+)) as well as under high Ca(2+) (5mM Mg(2+)/45 mM Ca(2+)). Under low Mg(2+), four Ca(2+) and three Mg(2+) occupy the same positions as in high-Mg(2+) structures. Conversely, under low Mg(2+), reexamination of the structure of Gla domain of activated Protein C (APC) complexed with soluble endothelial Protein C receptor (sEPCR) has position 4 occupied by Ca(2+) and positions 1 and 7 by Mg(2+). Nonetheless, in direct binding experiments, Mg(2+) replaced three Ca(2+) sites in the unliganded Protein C or APC. Further, the high-Ca(2+) condition was necessary to replace Mg4 in the FVIIa/soluble TF structure. In biological studies, Mg(2+) enhanced phospholipid binding to FVIIa and APC at physiological Ca(2+). Additionally, Mg(2+) potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF and inactivation of activated factor V by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low-Mg(2+) condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg(2+) and Ca(2+) act in concert to promote coagulation and anticoagulation.
Assuntos
Ácido 1-Carboxiglutâmico/química , Cálcio/metabolismo , Fator VIIa/química , Fator VIIa/metabolismo , Magnésio/metabolismo , Proteína C/química , Proteína C/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Alzheimer's disease (AD) is an irreversible neurodegenerative disorder with symptoms of confusion, memory loss, and mood swings. The beta-amyloid peptide, with 39-42 amino acid residues (BAP), plays a significant role in the development of AD. Although there is no cure for AD, it can be managed with available drugs to some degree. Several studies have revealed that natural antioxidants, such as vitamin E, vitamin C and beta-carotene, may help in scavenging free radicals generated during the initiation and progression of this disease. Therefore, there has been considerable interest in plant phytochemicals with antioxidant property as potential agents to prevent the progression of AD. Our earlier investigations of the Withania somnifera fruit afforded lipid peroxidation inhibitory withanamides that are more potent than the commercial antioxidants. In this study, we have tested two major withanamides A (WA) and C (WC) for their ability to protect the PC-12 cells, rat neuronal cells, from beta-amyloid induced cell damage. The cell death caused by beta-amyloid was negated by withanamide treatment. Molecular modeling studies showed that withanamides A and C uniquely bind to the active motif of beta-amyloid (25-35) and suggest that withanamides have the ability to prevent the fibril formation. Further understanding of the mechanism of action and in vivo efficacy of these withanamides may facilitate its development as a prophylaxis.
Assuntos
Doença de Alzheimer/prevenção & controle , Amidas/farmacologia , Peptídeos beta-Amiloides/efeitos adversos , Dissacarídeos/farmacologia , Indóis/farmacologia , Fragmentos de Peptídeos/efeitos adversos , Withania/química , Doença de Alzheimer/tratamento farmacológico , Amidas/isolamento & purificação , Animais , Sobrevivência Celular , Dissacarídeos/isolamento & purificação , Frutas/química , Indóis/isolamento & purificação , Modelos Moleculares , Estrutura Molecular , Células PC12 , RatosRESUMO
Phenol sulfotransferases (SULTs), which normally bind 3'-phosphoadenosine-5'-phosphosulfate as the donor substrate, are inhibited by CoA and its thioesters. Here, we report that inhibition of bovine SULT1A1 by CoA is time-dependent at neutral pH under non-reducing conditions. The rates of inactivation by CoA indicate an initial reversible SULT:CoA complex with a dissociation constant of 5.7 microM and an inactivation rate constant of 0.07 min(-1). Titrations with CoA and prolonged incubations reveal that inactivation of the dimeric enzyme is stoichiometric, consistent with the observation of complete conversion of the protein to a slightly decreased electrophoretic mobility. Both activity and normal electrophoretic migration are restored by 2-mercaptoethanol. Mutagenesis demonstrated that Cys168 is the site of CoA adduction, and a consistent model was constructed that reveals a new SULT molecular dynamic. Cysteine reaction kinetics with Ellman's reagent revealed a PAPS-induced structural change consistent with the model that accounts for binding of CoA.
Assuntos
Arilsulfotransferase/química , Coenzima A/química , Sulfetos/química , Animais , Arilsulfotransferase/antagonistas & inibidores , Arilsulfotransferase/genética , Bovinos , Cisteína/química , Cisteína/genética , Ativação Enzimática , Concentração de Íons de Hidrogênio , Cinética , Mercaptoetanol/química , Modelos Moleculares , MutaçãoRESUMO
Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.
Assuntos
Fator VIIa/química , Tromboplastina/química , Benzamidinas/química , Catálise , Fator VIIa/metabolismo , Humanos , Modelos Moleculares , Mapeamento de Peptídeos , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Tromboplastina/metabolismoRESUMO
The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC with K(d) values of approximately 24 mm in the absence and approximately 6 mm in the presence of Ca2+. Sodium-occupied APC has approximately 100-fold increased catalytic efficiency ( approximately 4-fold decrease in K(m) and approximately 25-fold increase in k(cat)) in hydrolyzing S-2288 (H-d-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases this k(cat) slightly ( approximately 1.2-fold). Ca2+ binds to the protease domain of APC with K(d) values of approximately 438 microm in the absence and approximately 105 microm in the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K(m) but increases the k(cat) approximately 10-fold, and Na+ further increases this k(cat) approximately 3-fold and decreases the K(m) value approximately 3.7-fold. In agreement with the K(m) data, sodium-occupied APC has approximately 4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity.