Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation.
The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.
Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan 710, Republic of China.
D H
DH
Wu
G Y
GY
Shi
W J
WJ
Chuang
J M
JM
Hsu
K C
KC
Young
C W
CW
Chang
H L
HL
Wu
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D010958:Plasminogen / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D010958:Plasminogen; D013300:Streptokinase