Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance

Abstract

Streptococcus equisirnilis 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 plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al ., 1996, Protein Sci 5693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46°C and its stability is largely independent of the presence of the other domains. The hightemperatwe transition in intact SK (at approximately 63 "C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (AI and A2) adopt unstructured conformations when separated. AI binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

Role of the amino‐terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides

“…Although entirely speculative at present, the fact that the Kplg values of both native SK-HPG and SK56-414-HPG actually decrease by nearly twofold in the presence of SK16-59 (Table 2) lends support to this possibility. However, additional factors, such as stabilization of the activator complex by the N-terminal region (as proposed by Conejero-Lara et al, 1996;Parrado et al, 1996), or its ability to compensate for the loss of this region from the activator complex and the consequent loss of activity (Siefring & Castellino, 1976) may also play a role in the twofold superactivation phenomenon, particularly if the presence of an excess of the externally added fragment allows, at saturation, a level of PC activator activity to be attained that would not be possible in even the native SK-HPG complex because of the degradation of this region.…”

Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains

“…SK has been shown to be composed of three structurally similar domains (termed ␣, ␤, and ␥), separated by random coils and small, flexible regions at the amino and carboxyl termini (5,7,8). The recently solved crystal structure of the catalytic domain of HPN complexed with SK strongly indicates how SK might modulate the substrate specificity of HPN by providing a "valley" or cleft in which the macromolecular substrate can dock through protein-protein interactions, thus positioning the scissile peptide bond optimally for cleavage by the HPN active site, thereby conferring a narrow substrate preference onto an otherwise "indiscriminate" active center.…”

“…Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK⅐HPN displays a very narrow substrate specificity (4). The structural basis of the conversion of the broadly specific serine protease HPN to a highly substrate-specific protease, once complexed with the "cofactor" SK, with exclusive propensity for acting on the target scissile peptide bond in HPG has been the subject of active investigations with both fundamental and applied implications (5)(6)(7)(8)(9)(10)(11)(12).…”

“…Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK⅐HPN displays a very narrow substrate specificity (4). The structural basis of the conversion of the broadly specific serine protease HPN to a highly substrate-specific protease, once complexed with the "cofactor" SK, with exclusive propensity for acting on the target scissile peptide bond in HPG has been the subject of active investigations with both fundamental and applied implications (5-12).SK has been shown to be composed of three structurally similar domains (termed ␣, ␤, and ␥), separated by random coils and small, flexible regions at the amino and carboxyl termini (5,7,8). The recently solved crystal structure of the catalytic domain of HPN complexed with SK strongly indicates how SK might modulate the substrate specificity of HPN by providing a "valley" or cleft in which the macromolecular substrate can dock through protein-protein interactions, thus positioning the scissile peptide bond optimally for cleavage by the HPN active site, thereby conferring a narrow substrate preference onto an otherwise "indiscriminate" active center.…”

Domain Truncation Studies Reveal That the Streptokinase-Plasmin Activator Complex Utilizes Long Range Protein-Protein Interactions with Macromolecular Substrate to Maximize Catalytic Turnover

“…It consists of a single polypeptide chain of 414 residues and is organized into three structurally similar, independently folding domains (termed ␣, ␤, and ␥ in order from N to C terminus of the polypeptide) that are separated by coiled coils and small flexible regions at the two ends (3)(4)(5). Like several other well known thrombolytic proteins, such as urokinase and tissue-plasminogen activator, SK exerts its effects through the conversion of human plasminogen (HPG) to its proteolytically active form, plasmin (HPN).…”