scholarly journals Highly conserved residue arginine-15 is required for the Ca2+-dependent properties of the γ-carboxyglutamic acid domain of human anticoagulation Protein C and activated Protein C

1997 ◽  
Vol 322 (1) ◽  
pp. 309-315 ◽  
Author(s):  
Abraham THARIATH ◽  
Francis J. CASTELLINO
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Wild Type ◽  
Phospholipid Binding ◽  
Critical Residue ◽  
Carboxyglutamic Acid

The function of the rigidly conserved amino acid residue R15 in the Ca2+/phospholipid-dependent properties of the γ-carboxyglutamic acid (Gla)-containing domain (GD) of human Protein C (PC) were investigated through site-directed mutagenesis strategies. A series of recombinant (r) mutants, namely r-[R15K]PC, r-[R15H]PC, r-[R15L]PC, and r-[R15W]PC, were constructed, expressed and purified, and their relevant properties investigated. As revealed by intrinsic fluorescence analysis, all of the variant proteins underwent Ca2+-dependent structural transitions. Nonetheless, they displayed altered binding properties to acidic phospholipid vesicles, and also did not interact with a monoclonal antibody specific for the type of Ca2+-dependent conformation of the GD that characterizes the wild-type protein. On conversion into their activated forms, these variant enzymes possessed less than 10% of the ex vivo plasma anticoagulant activity of wild-type r-PC. Similar activities were found when the r-active PC mutants were assayed directly for inactivation of factor Va and factor VIII, in the complete prothrombinase and tenase complexes respectively. We conclude that R15 is a critical residue in allowing the GD of PC, and probably of other proteins of this class, to adopt a Ca2+-dependent conformation that allows functional phospholipid binding, thus explaining the strict conservation of this amino acid residue in GD modules of various proteins. As a result of an analysis of structural models of the Ca2+ŐGD complex of PC, it is postulated that hydrogen bonds between the side chain of R15 and the functionally important Gla16 residue, as well as between the side chain of R15 and the carbonyl oxygen in the peptide bond of H10, are critical for adoption of a Ca2+-dependent conformation of the GD that allows functional phospholipid binding.

Properties of a Recombinant Chimeric Protein in which the gamma-Carboxyglutamic Acid and Helical Stack Domains of Human Anticoagulant Protein C Are Replaced by those of Human Coagulation Factor VII

1997 ◽  
Vol 77 (05) ◽  
pp. 0926-0933 ◽  
Author(s):  
Jie-Ping Geng ◽  
Francis J Castellino
Keyword(s):  
Metal Ion ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Chimeric Protein ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Wild Type ◽  
Coagulation Factor Vii ◽  
Carboxyglutamic Acid ◽  
Wild Type Counterpart

SummaryA chimeric cDNA, encoding residues 1-46 (the γ-carboxyglutamic acid module and its trailing helical stack) of human coagulant factor (f) VII, bound to residues 47-419 of human anticoagulant protein C (PC), was constructed and expressed. The resulting protein, r-[∆GD-HSPC/∇GD-HSfVII]PC, was properly processed with regard to signal/ propeptide release, cleavage of the K156R dipeptide, Gla and Hya contents, and the presence of glycosylation.The mutant protein displayed normal dependencies on Ca2+ for adoption of its metal ion-dependent conformation and for binding to acidic phospholipid vesicles. The chimera failed to recognize a monoclonal antibody (MAb) specific for the Ca2+-induced conformation of the Gla domain (GD) of PC, but did react with another MAb directed in part to the Ca2+-dependent conformation of the GD of fVII. Further, this chimeric protein possessed similar steady state constants as wild-type r-PC toward activation by thrombin and thrombin/thrombomodulin. The activated form of the chimera was very similar to that of its wild- type counterpart in its whole plasma anticoagulant activity, as well as its activity toward inactivation of coagulation factor VIII. The chimeric protein did not bind to the fVII cofactor, tissue factor, showing that the GD/HS domain region of fVII is insufficient for that particular interaction.The results demonstrate that the GD/HS of fVII, when present in the PC and APC background, serves to maintain the Ca2+/PL-related functions of these latter proteins, and suggest that the Ca2+ and PL- dependent interactions of the GD-HS of PC are sufficiently general in nature such that the GD-HS regions of other proteins of this type can satisfy most of the requirements of PC and APC. The data presented also offer support for the independent nature of the domain unit consisting of the GD/HS module.

scholarly journals UDP-glucose:anthocyanidin 3-O-glucoside-2”-O-glucosyltransferase catalyzes further glycosylation of anthocyanins in purple Ipomoea batatas

2018 ◽  
Author(s):  
Hongxia Wang ◽  
Chengyuan Wang ◽  
Weijuan Fan ◽  
Jun Yang ◽  
Ingo Appelhagen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Ipomoea Batatas ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Anthocyanin Accumulation ◽  
Wild Type ◽  
Different Tissues

AbstractGlycosylation contributes to the diversity and stability of anthocyanins in plants. The process is catalyzed by various glucosyltransferases using different anthocyanidin aglycones and glycosyl donors. An anthocyanidin 3-O-glucoside-2”-O-glucosyltransferase (3GGT) from purple sweetpotato (cv. Ayamurasaki) served for the catalytic conversion of anthocyanidin 3-O-glucoside into anthocyanidin 3-O-sophoroside, which is functionally different from the 3GGT ortholog of Arabidopsis. The phylogenetic analysis indicates regioselectivity of 3GGT using UDP-xylose or UDP-glucose as the glycosyl is divergent between Convolvulaceae and Arabidopsis. Homology-based protein modeling and site-directed mutagenesis of Ib3GGT and At3GGT suggested that the Thr-138 of Ib3GGT is a key amino acid residue for UDP-glucose recognition and plays a major role in sugar donor selectivity. The wild type and ugt79b1 mutants of Arabidopsis plants overexpressing Ib3GGT produced the new component cyanidin 3-O-sophoroside. Moreover, Ib3GGT expression was associated with anthocyanin accumulation in different tissues during Ayamurasaki plant development and was regulated by the transcription factor IbMYB1. The localization assay of Ib3GGT showed that further glycosylation occurs in the cytosol and not endoplasmic reticulum. The present study revealed the function of Ib3GGT in further glycosylation of anthocyanins and its Thr-138 is the key amino acid residue for UDP-glucose recognition.

scholarly journals Effect of roasting temperature on lipid and protein oxidation and amino acid residue side chain modification of beef patties

RSC Advances ◽  
2021 ◽  
Vol 11 (35) ◽  
pp. 21629-21641
Author(s):  
Chao Xia ◽  
Pingping Wen ◽  
Yaming Yuan ◽  
Xiaofan Yu ◽  
Yijing Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Protein Oxidation ◽  
Relative Number ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Beef Patties ◽  
Lipid And Protein Oxidation ◽  
Roasting Temperature

The relative number of peptides modified by the amino acid residues of actin from raw beef patties and those cooked at different roasting temperatures.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals The Role of Arg13 in Protein Phosphatase M tPphA from Thermosynechococcus elongatus

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Jiyong Su ◽  
Karl Forchhammer
Keyword(s):  
Amino Acid ◽  
Protein Phosphatase ◽  
Arginine Residue ◽  
Side Chain ◽  
Wild Type ◽  
Catalytic Center ◽  
Nitrophenyl Phosphate ◽  
Reduced Activity ◽  
Amino Acid Variants

A highly conserved arginine residue is close to the catalytic center of PPM/PP2C-type protein phosphatases. Different crystal structures of PPM/PP2C homologues revealed that the guanidinium side chain of this arginine residue can adopt variable conformations and may bind ligands, suggesting an important role of this residue during catalysis. In this paper, we randomly mutated Arginine 13 of tPphA, a PPM/PP2C-type phosphatase from Thermosynechococcus elongatus, and obtained 18 different amino acid variants. The generated variants were tested towards p-nitrophenyl phosphate and various phosphopeptides. Towards p-nitrophenyl phosphate as substrate, twelve variants showed 3–7 times higher Km values than wild-type tPphA and four variants (R13D, R13F, R13L, and R13W) completely lost activity. Strikingly, these variants were still able to dephosphorylate phosphopeptides, although with strongly reduced activity. The specific inability of some Arg-13 variants to hydrolyze p-nitrophenyl phosphate highlights the importance of additional substrate interactions apart from the substrate phosphate for catalysis. The properties of the R13 variants indicate that this residue assists in substrate binding.

scholarly journals Specificity of activated human protein C

1985 ◽  
Vol 230 (2) ◽  
pp. 497-502 ◽  
Author(s):  
S R Stone ◽  
J Hofsteenge
Keyword(s):  
Amino Acid ◽  
Rate Constant ◽  
Amino Acid Residue ◽  
Protein C ◽  
Activated Protein C ◽  
Order Rate Constant ◽  
Human Protein ◽  
Functional Protein ◽  
Hydrophobic Residues ◽  
Apolar Residue

Peptide p-nitroanilide substrates and peptidylchloromethane inhibitors were used to examine the specificity of activated human Protein C. Substrates with arginine in the P1 position had the highest activity. The best substrates and inhibitors, as judged by the second-order rate constant for their interaction with the enzyme, had an apolar residue in the P2 position. In contrast with thrombin [Kettner & Shaw (1981) Methods Enzymol. 80, 826-842], activated Protein C was able to accommodate large hydrophobic residues such as phenylalanine and leucine in the P2 position. In the P3 position, the enzyme preferred an apolar D-amino acid residue. The results of the present study have also indicated a suitable substrate and inhibitor to be used in the assay of functional protein C and of thrombomodulin.

scholarly journals Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity

1989 ◽  
Vol 86 (17) ◽  
pp. 6577-6581 ◽  
Author(s):  
T W Seeley ◽  
L Grossman
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Excision Repair ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Side Chain ◽  
Uv Resistance ◽  
Wild Type ◽  
General Applicability ◽  
Repair Capacity

The Escherichia coli UvrB protein possesses an amino acid sequence motif common to many ATPases. The role of this motif in UvrB has been investigated by site-directed mutagenesis. Three UvrB mutants, with amino acid replacements at lysine-45, failed to confer UV resistance when tested in the UV-sensitive strain N364 (delta uvrB), while five other mutants constructed near this region of UvrB confer wild-type levels of UV resistance. Because even the conservative substitution of arginine for lysine-45 in UvrB results in failure to confer UV resistance, we believe we have identified an amino acid side chain in UvrB essential to nucleotide excision repair in E. coli. The properties of two purified mutant UvrB proteins, lysine-45 to alanine (K45A) and asparagine-51 to alanine (N51A), were analyzed in vitro. While the K45A mutant is fully defective in incision of UV-irradiated DNA, K45A is capable of interaction with UvrA in forming an ATP-dependent nucleoprotein complex. The K45A mutant, however, fails to activate the characteristic increase in ATPase activity observed with the wild-type UvrB in the presence of UvrA and DNA. From these results we conclude that there is a second nucleotide-dependent step in incision following initial complex formation, which is defective in the K45A mutant. This experimental approach may prove of general applicability in the study of function and mechanism of other ATPase motif proteins.

Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342

1989 ◽  
Vol 9 (4) ◽  
pp. 1406-1414
Author(s):  
A A McCracken ◽  
K B Kruse ◽  
J L Brown
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitor ◽  
Conformational Stability ◽  
Point Mutations ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Importance ◽  
Bridge Formation ◽  
Cos Cells

Human alpha-1-proteinase inhibitor (A1PI) deficiency, associated with the Z-variant A1PI (A1PI/Z) gene, results from defective secretion of the inhibitor from the liver. The A1PI/Z gene exhibits two point mutations which specify amino acid substitutions, Val-213 to Ala and Glu-342 to Lys. The functional importance of these substitutions in A1PI deficiency was investigated by studying the secretion of A1PI synthesized in COS cells transfected with A1PI genes altered by site-directed mutagenesis. This model system correctly duplicates the secretion defect seen in individuals homozygous for the A1PI/Z allele and shows that the substitution of Lys for Glu-342 alone causes defective secretion of A1PI. The substitution of Lys for Glu-342 eliminates the possibility for a salt bridge between residues 342 and 290, which may decrease the conformational stability of the molecule and thus account for the secretion defect. However, when we removed the potential to form a salt bridge from the wild-type inhibitor by changing Lys-290 to Glu (A1PI/SB-290Glu), secretion was not reduced to the 19% of normal level seen for A1PI/Z-342Lys; in fact, 75% of normal secretion was observed. When the potential for salt bridge formation was returned to A1PI/Z-342Lys by changing Lys-290 to Glu, only 46% of normal secretion was seen. These data indicate that the amino acid substitution at position 342, rather than the potential to form the 290-342 salt bridge, is the critical alteration leading to the defect in A1PI secretion.

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