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J Biol Chem, Vol. 273, Issue 35, 22346-22350, August 28, 1998

NH₂-terminal Structural Motifs in Staphylokinase Required for Plasminogen Activation

Bernhard Schlott, Karl-Heinz Gührs, Manfred Hartmann, Anja Röcker, and Désiré Collen^¶

From the  Institute for Molecular Biotechnology, Jena, 07745 Germany and the ^¶ Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, B-3000 Leuven, Belgium

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys¹⁰-Lys¹¹ peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH₂-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH₂-terminal region Lys¹¹-Gly-Asp-Asp-Ala-Ser¹⁶-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis.

Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH₂-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH₂-terminal amino acid substituted with Lys-generated derivatives from which the NH₂-terminal initiation Met was no longer removed, yielding inactive ( 10%) Sak42DN11(M),G12K, active (>50%) Sak42DN14(M),A15K and Sak42DN15(M),S16K, and inactive Sak42DN16(M),Y17K. Lys variants without NH₂-terminal Met, generated from fusion proteins in which a His₆ tag and a factor Xa recognition sequence were linked to the NH₂ terminus of the Sak variants, were indistinguishable from their NH₂-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DN11(M),G12K could be restored by additional substitution of both Asp¹³ and Asp¹⁴ with Asn, yielding active Sak42DN11(M),G12K, D13N, D14N, whereas substitution in Sak42DN16(M),Y17K of Phe¹⁸ and Glu¹⁹ with Asn yielded inactive Sak42DN16(M),Y17K,F18N,E19N.

These data, in combination with the recent finding that the 20 NH₂-terminal amino acids of Sak lack secondary structure, suggest that the NH₂-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH₂-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.

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