Author:

Keywords:

Cardiovascular System & Hematology

Abstract:

Staphylokinase (Sak), a 16-kDa profibrinolytic protein secreted by some strains of Staphylococcus aureus, forms a plasminogen activator complex with the serine protease domain of human plasmin (μPli). Unlike plasmin alone, the resulting 1:1 stoichiometric complex has the same functionality as tissue-type plasminogen activator (t-PA) and urokinase, i.e. cleavage of the Arg561-Val562 peptide bond of plasminogen. A comprehensive scanning mutagenesis program on Sak (27 mutants) and on uPli (25 mutants) has revealed the molecular determinants involved in the interface and the functionality of the activator complex. In the Sak molecule, the following amino acid residues were identified as critical for binding: K11, D14, Y24, M26, E46, Y44 and D69. Alaninereplacement at five other positions (V45, Y47, Y62, Y63 and W66) did not result in loss of binding but dramatically affected the plasminogen activation potential of the complex. Interestingly, on the 3D structure of Sak, these latter residues are located in the 2 sheet and the a helix where they form an aromatic cluster delineated by several of the amino acids critical for complex formation:E46, Y44, E69. The spatial position of Kl 1 and D14 is less defined due to the flexibility of the N-terminal region of Sak (residues 1 to 16). In uPli, seven amino acids were pointed out: N711, Y713, G718, R719, E724, R767 and K770. Mutation at each of these positions leads to variants endowed with full amidolytic activity but three of them (G718E, R719A, R767A) showed a strongly impaired plasminogen activating ability when mixed with Sak. On a homology-modeled μPli structure, the critical amino acids are located on two loops, L8 and L11, in close proximity of the catalytic triad. In an attempt to dock Sak on uPli, compensatory mutations on Sak were identified for restoring binding ability to uPli G718E and to (jPli R767A. Sak mutations were searched by panning a randomized Sak library displayed on phage on immobilized jjPli mutants. Proximity relationships were deduced between 0718 on uPli and the β2 sheet on one hand, and between R767 and the extended N-terminal end on the other hand. A 3D model of the Sak:μPli complex was created in agreement with the experimental data. This model confirms previous observations wherein the Sak:Pli complex could accomodate a Kunitz inhibitor but not a Kazal-type one because of sterical hindrance. Finally, this model also allows to formulate a role for the aromatic cluster of Sak in the mechanism of plasminogen activation and to deduce structural relationships with t-PA and urokinase.