TY - JOUR
T1 - Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation
AU - Liu, Lin
AU - Sazonova, Irina Yurievna
AU - Turner, Ryan B.
AU - Chowdhry, Shakeel A.
AU - Tsai, Judy
AU - Houng, Aiilyan K.
AU - Reed, Guy L.
PY - 2000/12/1
Y1 - 2000/12/1
N2 - The NH2 terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not arnidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKA59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKΔ59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular inter-actions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.
AB - The NH2 terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not arnidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKA59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKΔ59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular inter-actions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.
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U2 - 10.1074/jbc.M003963200
DO - 10.1074/jbc.M003963200
M3 - Article
C2 - 10961989
AN - SCOPUS:0034529160
SN - 0021-9258
VL - 275
SP - 37686
EP - 37691
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 48
ER -