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DOI: 10.1055/s-0038-1648974
In Vitro Mutagenesis Study of Two Critical Glutamic Acids in the Calcium Binding Loop of the Factor IX Heavy Chain
Publikationsverlauf
Received 11. Mai 1994
Accepted after resubmission 02. September 1994
Publikationsdatum:
06. Juli 2018 (online)
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Summary
We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium’s absence, factor IXGlu235Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245LyS’s conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium. We conclude that glutamic acids 235 and 245 are, as predicted, involved in the calcium binding site in the factor IX catalytic domain, and that the correct conformation of this calcium binding loop is essential for maintaining the active site pocket.
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References
- 1 Thompson A. Structure, function and molecular defects of factor IX. Blood 1986; 67: 565-572
- 2 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16
- 3 Wildgoose P, Foster D, Schipdt J, Wiberg F, Birktoft JJ, Petersen LC. Identification of a calcium binding site in the protease domain blood coagulation factor VII: evidence for its role in factor VH-tissue factor interaction. Biochemistry 1993; 32: 114-119
- 4 Bajaj SP, Sabharwal AK, Gorka J, Birktoft A. Antibody-probed conformational transitions in the protease domain of human factor IX upon calcium binding and zymogen activation: putative high-affinity Ca2+-binding site in the protease domain. Proc Natl Acad Sci USA 1992; 89: 152-156
- 5 Persson E, Hogg PJ, Stenflo J. Effects of Ca2++ binding on the protease module of factor Xa and its interaction with factor Va. JBC 1993; 268: 22531-22539
- 6 Rezaie A, Mather T, Sussman F, Esmon C. Mutation of Glu-80-lys results in a protein C mutant that no longer requires Ca2++ for rapid activation by the thrombin-thrombomodulin complex. JBC 1994; 269: 3151-3154
- 7 Bode W, Mayr I, Baumann U, Huber R, Stone SR, Hofsteenge J. The refined 1.0 A crystal structure of human a-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J 1989; 8: 3467-75
- 8 Giannelli F, Green PM, High KA, Sommer S, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Brownlee GG. Haemophilia B: database of point mutations and short additions and deletions -fourth edition. Nucleic Acids Res 1993; 21: 3075-3087
- 9 Sarkar G, Koeberl DD, Sommer SS. Direct sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species. Genomics 1990; 6: 133-143
- 10 Frazier D, Smith K, Cheung W-F, Ware J, Lin S-W, Thompson A, Reisner H, Bajaj P, Stafford D. Mapping of monoclonal antibodies to human factor IX. Blood 1989; 7: 971-977
- 11 Bajaj SP, Rapaport SI, Maki SL. A monoclonal antibody to factor IX that inhibits the factor VIII: Ca potentiation of factor X activation. J Biol Chem 1985; 260: 11574-11580
- 12 Hamaguchi N, Charifson P, Pedersen L, Brayer G, Smith K, Stafford D. Expression and characterization of human factor IX. J Biol Chem 1991; 266: 15213-15220
- 13 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
- 14 Monroe DM, Sherrill GB, Roberts HR. Use of p-aminobenzamidine to monitor activation of trypsin-like serine proteases. Anal Biochem 1988; 172: 427-435
- 15 Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C, Alagona G, Profeta Jr S, Weiner P. A new force field for molecular mechanical simulation of nucleic acids and proteins. J Am Chem Soc 1984; 106: 765-784
- 16 Geddes VA, LeBonniec BF, Louie GV, Brayer GD, Thompson AR, MacGillivray RTA. A moderate form of Hemophilia B is caused by a novel mutation in theprotease domain of factor IXVancouvcr. J Biol Chem 1989; 264: 4689-4697
- 17 Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 1983; 22: 2577-2637
- 18 Martin RB. Structural Chemistry of Calcium: Lanthanides as Probes. In: Calcium in Biology Spiro TG. ed Wiley-Interscience; New York, NY: 1983. pp 235-270
- 19 Sommerville LE, Thomas DD, Nelsestuen GL. Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1. J Biol Chem 1985; 260: 10444-10452
- 20 Schipdt J, Harrit N, Christensen U. Petersen LC. Two different Ca2+ ion binding sites in factor Vila and in des (1-38) factor Vila. FEBS Lett 1992; 306: 265-268