Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator

Fibrin-dependent plasminogen activation by tissue-type plasminogen activator (t-PA) is in part associated with the presence of the kringle 2 domain in t-PA, Within this kringle 2 domain a lysyl-binding site has been described, The plasminogen to plasmin conversion by urokinase-type plasminogen activator (u-PA), in contrast to that of t-PA, is not enhanced in the presence of fibrin, Within the u-PA kringle domain no lysyl-binding site is found, To study whether introduction of a lysyl-binding site in the u-PA kringle domain will make u-PA a fibrin-dependent plasminogen activator, three stretches of amino acid residues of the u-PA kringle domain (A(28)-Q(33), D-55-N-57 and G(67)-V-72) were substituted by three stretches of amino acids from the corresponding positions of the kringle 2 domain of t-PA (M(28)-K-33, D-55-D-57 and N-67-W-72). These changes resulted in the creation of the lysyl-binding site consensus of the kringle 2 domain (K-33, D-55, D-57, W-62 and W-72) in the u-PA kringle, However, the resulting u-PA mutant did not interact with lysyl-Sepharose, nor did it display fibrin-enhanced plasminogen activation in the presence of soluble fibrin mimic, When the kringle domain of u-PA was replaced by the kringle 2 domain of t-PA, similar results were obtained, The hybrid protein hardly interacted with lysyl-Sepharose and the plasminogen activation was not enhanced in the presence of fibrin mimic, However, the N-terminal fragment isolated from this hybrid molecule (consisting of growth factor domain and kringle 2 domain) did interact with lysyl-Sepharose, suggesting that in the hybrid molecule a functional lysyl-binding site is present but not operational, Indeed, lysine analogue (E-aminocaproic acid) sensitive binding of isolated t-PA kringle 2 domain to u-PA could be observed, The modified u-PA kringle, the wild type u-PA kringle and the kringle 2 of the u-PA hybrid were also placed N-terminal of the protease domain of t-PA, As expected, the t-PA mutant consisting of the kringle 2 domain and the protease domain bound to lysyl-Sepharose and showed fibrin-dependent plasminogen activation, Further, the hybrid molecule consisting of the u-PA kringle placed N-terminal of the t-PA protease domain did not display these features, Introduction of the modified u-PA kringle N-terminal of the t-PA protease domain resulted in a very weak interaction with lysyl-Sepharose, Despite the high overall similarity in primary structure of the modified u-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogen activation of this hybrid molecule was observed, The above-mentioned results question the concept that the structural auto-nomous domains within hybrid plasminogen activators t-PA and u-PA function as autonomous domains and suggest that interactions between the kringle and the protease domain in hybrid molecules strongly influences their functional features.