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

Jayeeta Dhar,Abhay H Pande,Vasudha Sundram,Jagpreet S Nanda,Shekhar C Mande,Girish Sahni

doi:10.1074/jbc.m108422200

Jayeeta Dhar, Abhay H Pande + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m108422200

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Abstract

The selective deletion of a discrete surface-exposed epitope (residues 254-262; 250-loop) in the beta domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK(del254-262)). A kinetic analysis of SK(del254-262) revealed that its low HPG activator activity arose from a 5-6-fold increase in K(m) for HPG as substrate, with little alteration in k(cat) rates. This increase in the K(m) for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK(del254-262). The involvement of kringles was further indicated by a hypersusceptibility of the SK(del254-262).plasmin activator complex to epsilon-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK.plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK(del254-262).HPG was reduced by about 3-fold in comparison with that of nSK.HPG . Overall, these observations identify the 250 loop in the beta domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK.HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process.

Highlights

Streptokinase (SK),1 a bacterial protein secreted by the Lancefield Group C ␤-hemolytic streptococci, is widely used as a thrombolytic agent in the treatment of various circulatory disorders, including myocardial infarction [1]
The recently solved crystal structure of the catalytic domain of HPG or plasmin (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, 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
Peptide walking studies in our laboratory had indicated that short peptides based on the primary structure of SK, those derived from selected regions in the ␣ and ␤ domains, displayed competitive inhibition for human plasminogen (HPG) activation by the preformed SK1⁄7HPN complex under conditions where the 1:1 complexation of SK and HPN was essentially unaffected [11, 12] the crystal structure of SK complexed with microplasmin(ogen) [8], while providing a high degree of resolution of the residues involved in the SK1⁄7␮PN complexation, yielded few unambiguous insights regarding the interactions engendered between the activator complex and substrate HPG

Summary

EXPERIMENTAL PROCEDURES

Glu-plasminogen was either purchased from Roche Diagnostics Inc. or purified from human plasma by affinity chromatography [19]. In experiments where EACA was used to examine its effect on SK-HPG interaction, the binary complex was formed between ligate nSK/SKdel254262 and immobilized HPG in binding buffer (as described above). Ternary Interaction Analysis—Resonant mirror technology-based biosensor was used to measure the rate and equilibrium dissociation constants describing interactions between soluble ligate (PG, ␮PG, or K1–5) and nSK/SKdel254262 complexed with immobilized HPG, a situation simulating substrate binding to binary complex and hereafter referred to as ternary interaction. For the analysis of these data, the binding extent in buffer alone was taken as 100%, and varying extents of formation of ternary complexes at equilibrium were plotted as a function of EACA concentration to obtain EACAdependent binding isotherms for nSK and SKdel254262. The final spectrum analyzed was an average of 10 scans [31]

Modeling Studies

RESULTS AND DISCUSSION

Activator protein

Methods