Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation.
The bacterial protein streptokinase (SK) activates human plasminogen (Pg) into the fibrinolytic protease plasmin (Pm). Roughly 40 residues from the SK C-terminal domain are mobile in the crystal structure of SK complexed with the catalytic domain of Pm, and the functions of this C-tail remain elusive. To better define its roles in Pg activation, we constructed and characterized three C-terminal truncation mutants containing SK residues 1-378, 1-386, and 1-401, respectively. They exhibit gradually reduced amidolytic activity and Pg-activator activity, as well as marginally decreased binding affinity toward Pg, as more of the C-terminus is deleted. As compared with full-length SK, the shortest construct, SK(1-378), exhibits an 80% decrease in amidolytic activity (k(cat)/K(M)), an 80% decrease in Pg-activator activity, and a 30% increase in the dissociation constant toward the Pg catalytic domain. The C-terminal truncation mutations did not attenuate the resistance of the SK-Pm complex to alpha(2)-antiplasmin. Attempts at using a purified C-tail peptide to rescue the activity loss of the truncation mutants failed, suggesting that the integrity of the SK C-terminal peptide is important for the full function of SK.
Zhai
Peng
P
Crystallography Research Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA.
Wakeham
Nancy
N
Loy
Jeffrey A
JA
Zhang
Xuejun C
XC
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D010958:Plasminogen / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics; D013300:Streptokinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity / Q000235:genetics; D000979:alpha-2-Antiplasmin / Q000737:chemistry
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; C111879:microplasminogen protein, human; D010958:Plasminogen; D013300:Streptokinase; D010960:Plasminogen Activators