المؤلفون: Enrico Di Cera, Likui Yang, Alireza R. Rezaie, Swati Prasad

المصدر: Journal of Biological Chemistry. 279:38519-38524

مصطلحات موضوعية: Protein Conformation, Stereochemistry, medicine.medical_treatment, Mutant, Plasma protein binding, Biochemistry, Divalent, Protein structure, Mutant protein, Cations, medicine, Animals, Chymotrypsin, Binding site, Molecular Biology, Ions, chemistry.chemical_classification, Binding Sites, Protease, Dose-Response Relationship, Drug, biology, Chemistry, Sodium, Thrombin, Cell Biology, Molecular biology, Recombinant Proteins, Protein Structure, Tertiary, Kinetics, Spectrometry, Fluorescence, Mutation, biology.protein, Calcium, Cattle, Peptides, 1-Carboxyglutamic Acid, Protein Binding, Protein C

الوصف: Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a936efafa5808d0ee52125b3ad448ff7Test
https://doi.org/10.1074/jbc.m407304200Test

المؤلفون: Kota N. Gopalakrishna, Likui Yang, Alireza R. Rezaie, Chandrashekhara Manithody

المصدر: Protein expression and purification. 50(2)

مصطلحات موضوعية: Models, Molecular, Proteases, Stereochemistry, Protein Conformation, medicine.medical_treatment, Genetic Vectors, Catalysis, Factor IXa, chemistry.chemical_compound, Thrombin, medicine, Humans, Binding site, Factor VIIIa, Factor IX, Chymotrypsin, Protease, Alanine, biology, Factor X, Sodium, Recombinant Proteins, Neoplasm Proteins, Enzyme Activation, Cysteine Endopeptidases, Biochemistry, chemistry, Mutation, biology.protein, Calcium, Biotechnology, medicine.drug, Plasmids, Protein Binding

الوصف: Recent studies have indicated that the loop harboring the S1 specificity site (residues 185–189 in chymotrypsin numbering) of coagulation proteases has several charged residues with important structural and functional roles for the catalytic activity of these proteases. This loop is allosterically linked to the Na + -binding site in both factor Xa and thrombin. There are three candidate residues (His-185, Glu-186, and Arg-188) on this loop of factor IXa (fIXa) whose side chains can influence the Na + binding and the catalytic function of the protease in the intrinsic Xase complex. In this study, we developed a novel expression/purification vector system, substituted all three residues of factor IX individually with Ala, and expressed the mutant zymogens in mammalian cells. Following activation, all three fIXa mutants exhibited normal activity towards a fIXa-specific chromogenic substrate in the presence of Ca 2+ with no obvious requirement for Na + in the reaction. Furthermore, all three mutants interacted with factor VIIIa with near normal affinity and catalyzed the activation of factor X in the intrinsic Xase complex with a normal catalytic efficiency. These results suggest that, unlike thrombin and factor Xa, the charged residues of this loop do not play a functional role in modulating the catalytic function of fIXa in the intrinsic Xase complex.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::842d1efcea35e312c54dc2f05431d2beTest
https://pubmed.ncbi.nlm.nih.gov/16829135Test

المؤلفون: Likui Yang, Alireza R. Rezaie, Chandrashekhara Manithody

مصطلحات موضوعية: Models, Molecular, Time Factors, Stereochemistry, Protein Conformation, Thrombomodulin, Beta sheet, Plasma protein binding, Antiparallel (biochemistry), Arginine, Models, Biological, Antithrombins, Protein Structure, Secondary, Cell Line, Thrombin, Protein structure, Catalytic Domain, medicine, Humans, Binding site, Edetic Acid, Multidisciplinary, Alanine, Binding Sites, Dose-Response Relationship, Drug, Chemistry, Biological Sciences, Recombinant Proteins, Kinetics, Mutation, Calcium, Electrophoresis, Polyacrylamide Gel, Receptors, Thrombin, Protein C, medicine.drug, Protein Binding

الوصف: The binding of Ca 2+ to the 70–80 loop of protein C inhibits protein C activation by thrombin in the absence of thrombomodulin (TM), but the metal ion is required for activation in the presence of TM. Structural data suggests that the 70–80 loop is located between two antiparallel β strands comprised of residues 64–69 and 81–91 on the protease domain of protein C. To test the hypothesis that a salt-bridge/hydrogen bond interaction between Arg-67 of the former strand and Asp-82 of the latter strand modulates the unique Ca 2+ -binding properties of protein C, we engineered a disulfide bond between the two strands by substituting both Arg-67 and Asp-82 with Cys residues. The activation of this mutant was enhanced 40- to 50-fold independent of TM and Ca 2+ . Furthermore, the Arg-67 to Ala mutant of protein C was activated in the absence of TM by the Arg-35 to Glu mutant of thrombin with the same efficiency as wild-type protein C by wild-type thrombin–TM complex. These results suggest that TM functions by alleviating the Ca 2+ -dependent inhibitory interactions of Arg-67 of protein C and Arg-35 of thrombin.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4d394e5220abb2ef704c36e9d636bccaTest
https://europepmc.org/articles/PMC1347983Test/

المؤلفون: Alireza R. Rezaie

المصدر: The Journal of biological chemistry. 275(5)

مصطلحات موضوعية: Stereochemistry, Protein Conformation, Molecular Sequence Data, Plasma protein binding, Biochemistry, Cofactor, Thrombin, Protein structure, Prothrombinase, medicine, Humans, Binding site, Molecular Biology, Binding Sites, biology, Chemistry, Heparin, Antithrombin, Cell Biology, Recombinant Proteins, Factor Va, Factor Xa, biology.protein, Calcium, medicine.drug, Protein Binding

الوصف: We recently demonstrated that a template mechanism makes a significant contribution to the heparin-accelerated inactivation of factor Xa (FXa) by antithrombin at physiologic Ca(2+), suggesting that FXa has a potential heparin-binding site. Structural data indicate that 7 of the 11 basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in FXa. These residues, Arg(93), Lys(96), Arg(125), Arg(165), Lys(169), Lys(236), and Arg(240) were substituted with Ala in separate constructs in Gla domainless forms. It was found that all derivatives cleave Spectrozyme FXa with similar catalytic efficiencies. Antithrombin inactivated FXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of heparin, however, k(2) with certain mutants were impaired up to 25-fold. Moreover, these mutants bound to heparin-Sepharose with lower affinities. Heparin concentration dependence of the inactivation revealed that only the template portion of the cofactor effect of heparin was affected by the mutagenesis. The order of importance of these residues for binding heparin was as follows: Arg(240) > Lys(236) > Lys(169) > Arg(165) > Lys(96) > Arg(93) >/= Arg(125). Interestingly, further study suggested that certain basic residues of this site, particularly Arg(165) and Lys(169), play key roles in factor Va and/or prothrombin recognition by FXa in prothrombinase.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::96a8dfc293e538fc7d4063043363128eTest
https://pubmed.ncbi.nlm.nih.gov/10652320Test