1.8 Å crystal structure of staphylokinase: a promising thrombolytic agent

A. Rabijns, H.L. De Bondt & C. De Ranter

Laboratory for Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, E. Van Evenstraat 4, B-3000 Leuven, Belgium

Keywords: Protein, Structure, Staphylokinase, Thrombolytic agent

Staphylokinase, a 136 amino acids protein produced by certain Staphylococcus aureus strains has been shown to be a very potent plasminogen activator (1). It has recently emerged as a potent and highly fibrin-specific thrombolytic agent for the treatment of patients with acute myocardial infarction or peripheral arterial occlusions (2,3). Like streptokinase, which is a routinely used thrombolytic agent, staphylokinase is not an enzyme as it does not show any proteolytic activity by itself. Rather it forms a 1:1 complex with plasmin, which in turn activates other plasminogen molecules (4). In contrast to streptokinase, however, staphylokinase is able to induce lysis in a plasma milieu without causing systemic plasminogen activation and marked fibrinogen breakdown, thereby decreasing the risk of severe bleeding. Hence, staphylokinase might well be a promising alternative to streptokinase for the treatment of acute myocardial infarction and in future it may become the thrombolytic agent of choice. However, the mechanism of plasminogen activation by staphylokinase is still not fully understood and, being a heterologous protein of bacterial origin, it induces neutralising antibody formation after administration in humans (5). In an effort to further understand the mechanism of plasminogen activation by staphylokinase and to provide a basis for rational epitope mapping, we have solved the crystal structure of recombinant staphylokinase (SakSTAR variant) to a resolution of 1.8 Å.

Recombinant staphylokinase was crystallised in a hanging drop vapour diffusion set-up using a precipitant solution containing MgCl₂, Tris buffer pH 8.5 and PEG 4K. The crystals grow in the C2 space group with a = 60.6 Å, b = 43.7 Å, c = 54.3 Å and b = 115.6° and the MIRAS method was used to phase the data.

Staphylokinase is composed of a mixed five-stranded b-sheet which is packed on a single 12 residue a-helix. Using the notation of Richardson, the topology of the sheet is +1, -3x, -1, 2x with two right-handed crossovers each containing a b-strand that, together, form a two-stranded anti-parallel b-sheet. Starting from residue 16 staphylokinase folds into a compact, flattened and somewhat elongated structure. Residues 16-20 extend from the compact ellipsoid of the molecule and they point towards a large solvent-filled intermolecular cavity in which the rest of the N-terminus (residues 1-15) seems to be disordered. Biochemical studies of staphylokinase have uncovered portions of the molecule that are functionally important. It was e.g. shown that amino acids 46, 50, 65 and 69 are essential for complex formation with plasmin and for induction of active-site exposure in plasmin (6). Interestingly, we found that these regions map to the same side of the staphylokinase structure. Moreover, the N-terminus, which has previously been shown to be necessary for the activity of staphylokinase (6), is also situated on this side of the molecule (although it is disordered). This may imply that the complex formation of staphylokinase with plasmin occurs through this side of the molecule.

It is now generally accepted that most of the surface of a protein is potentially antigenic and that most antigenic sites are discontinuous and conformational in nature, i.e. composed of amino acids that are located on several loops brought close together by folding into the three-dimensional structures. This situation can also be seen in the staphylokinase structure. The analysis of a panel of 17 murine monoclonal antibodies against staphylokinase and a number of staphylokinase mutants has provided more detailed information regarding the antigenicity of this protein (7). Three non-overlapping immunodominant epitopes were recognised. Amino acids 74, 75 and 77 were found to constitute epitope 1, while epitope 3 is composed of amino acids 35, 38, 80 and 82. The residues constituting epitope 2 are not yet known. Although epitope 3 consists of amino acids that are distant in sequence, mapping of this mimotope on the three-dimensional structure of staphylokinase reveals that all its amino acids are brought together into one restricted patch which is located in the functionally less important back side of the protein. This opens perspectives for the future development of less immunogenic staphylokinase mutants. For epitope 1 the situation is less clear. Epitope 1 is situated on the front side of staphylokinase but is still quite distant from the residues that are necessary for plasminogen activation.

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