Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation.
The NH(2) 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 amidolysis (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 (rSKDelta59). 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 SKDelta59, 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 interactions 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.
Liu
L
L
Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Sazonova
I Y
IY
Turner
R B
RB
Chowdhry
S A
SA
Tsai
J
J
Houng
A K
AK
Reed
G L
GL
D001483:Base Sequence; D017931:DNA Primers; D005337:Fibrin / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D005353:Fibronectins; D011994:Recombinant Proteins; D005337:Fibrin; D010958:Plasminogen; D013300:Streptokinase; D007930:Leucine