THERMAL-STABILITY OF THE 3 DOMAINS OF STREPTOKINASE STUDIED BY CIRCULAR-DICHROISM AND NUCLEAR-MAGNETIC-RESONANCE

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plas minogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and t heir complex. We reported recently (Parrado J et al., 1996, Protein Sc i 5:693-704) that SK is composed of three structural domains (A, B, an d C) with a C-terminal tail that is relatively unstructured Here, we r eport thermal unfolding experiments, monitored by CD and NMR, using sa mples of intact SK, five isolated SK fragments, and two two-chain nonc ovalent complexes between complementary fragments of the protein. Thes e experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and int erdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individu al domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximatel y 46 degrees C and its stability is largely independent of the presenc e of the other domains. The high-temperature transition in intact SK ( at approximately 63 degrees C) corresponds to the unfolding of both do mains A and C. Thermal stability of domain C is significantly increase d by its isolation from the rest of the chain. By contrast, cleavage o f the Phe 63-Ala 64 peptide bond within domain A causes thermal destab ilization of this domain. The two resulting domain portions (A1 and A2 ) adopt unstructured conformations when separated. Al binds with high affinity to all fragments that contain the A2 portion, with a concomit ant restoration of the native-like fold of domain A. This result demon strates that the mechanism whereby A1 stimulates the plasminogen activ ator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.