Structural and functional basis of plasminogen activation by staphylokinase.
Staphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of microplasmin (microPli), the serine protease domain of plasmin. Sak binding on microPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on microPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed beta-sheet. Several Sak residues located within the unique alpha-helix and the beta2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:microPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of microPli, protrudes into a broad groove near the catalytic triad of microPli, thereby generating a competent binding pocket for micro-plasminogen (microPlg), which buries approximately 2500 A of the Sak:microPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:microPli:microPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.
Jespers
L
L
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.
Vanwetswinkel
S
S
Lijnen
H R
HR
Van Herzeele
N
N
Van Hoef
B
B
Demarsin
E
E
Collen
D
D
De Maeyer
M
M
D001435:Bacteriophages; D001665:Binding Sites; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005341:Fibrinolysin / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000494:pharmacology*; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000737:chemistry* / Q000494:pharmacology / Q000502:physiology*; D011480:Protease Inhibitors / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics; D013210:Staphylococcus / Q000821:virology
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus