Identification through Combinatorial Random and Rational Mutagenesis of a Substrate-interacting Exosite in the γ Domain of Streptokinase

Abstract

To identify new structure-function correlations in the ␥ domain of streptokinase, mutants were generated by error-prone random mutagenesis of the ␥ domain and its adjoining region in the ␤ domain followed by functional screening specifically for substrate plasminogen activation. Single-site mutants derived from various multipoint mutation clusters identified the importance of discrete residues in the ␥ domain that are important for substrate processing. Among the various residues, aspartate at position 328 was identified as critical for substrate human plasminogen activation through extensive mutagenesis of its side chain, namely D328R, D328H, D328N, and D328A. Other mutants found to be important in substrate plasminogen activation were, namely, R319H, N339S, K334A, K334E, and L335Q. When examined for their 1:1 interaction with human plasmin, these mutants were found to retain the nativelike high affinity for plasmin and also to generate amidolytic activity with partner plasminogen in a manner similar to wild type streptokinase. Moreover, cofactor activities of the mutants precomplexed with plasmin against microplasminogen as the substrate as well as in silico modeling studies suggested that the region 315-340 of the ␥ domain interacts with the serine protease domain of the macromolecular substrate. Overall, our results identify the presence of a substrate specific exosite in the ␥ domain of streptokinase.Streptokinase (SK), 3 secreted by several ␤-hemolytic group C (1, 2) strains of the genus Streptococcus, is a flexible multidomain protein (3,4). SK is often employed as a thrombolytic drug because it has the ability to activate human plasminogen (HPG), a zymogen, to its enzymatically active form, the serine protease, plasmin (HPN) (5, 6). However, unlike the physiological HPG activators such as tPA and urokinase, SK first forms a high affinity equimolar complex with HPG that results in the conformational activation of HPG to form SK⅐HPG* that specifically recruits substrate HPG and cleaves the Arg-561-Val-562 bond in substrate HPG molecules, thereby converting these into HPN (Pathway I, conformational activation pathway) (5, 6). The N-terminal Ile of SK has been demonstrated to play a critically important role in the activation of the "partner" after the formation of the 1:1 binary complex between SK and HPG (7-9). In addition, through several elegant studies on the mechanism of zymogen activation in the SK⅐HPG complex, it has been shown that HPN exhibits much higher affinity for SK compared with HPG, as a result of which an intermolecular exchange reaction takes place wherein the SK⅐HPN activates free substrate HPG molecules into HPN (10, 11). SK can also directly act as a co-factor for HPN, which upon complexation with HPN (12) (Pathway II, direct proteolytic activation pathway) virtually transforms the trypsin-like broad substrate specificity of HPN toward the cleavage of the scissile peptide bond, Arg-561-Val-562, in the incoming substrate HPG (12, 13).The mechanism by which SK alters the substrate specifi...

Functional differences between Streptococcus pyogenes cluster 1 and cluster 2b streptokinases are determined by their β‐domains

“…Numerous studies have been aimed at elucidating the function of each SK domain in hPg/hPm binding and activation. Various SK variants, including single and multiple domain constructs and point mutations [8][9][10][11][12][13][14][15][16][17][18][19], have been generated to attempt to clarify the functions of each of the SK domains in hPg binding and activation. Collectively, these studies generated a complex mixture of functional effects leading to a variety of different conclusions, but all of these studies agreed that there is some level of cooperation between the domains in hPg activation.…”

Multiple Exosites Distributed across the Three Domains of Streptokinase Co-Operate to Generate High Catalytic Rates in the Streptokinase–Plasmin Activator Complex

“…Remarkably, none of these sites have been shown to confer any substantial enzyme–substrate affinity, but (as their mutations show) these are nevertheless important in generating high catalytic rates for plasminogen activation by the SK·HPG complex. In contrast, the 250 loop in the β domain had earlier been shown to selectively enhance the affinity of the SK·HPG activator complex with substrate HPG through kringle mediated interactions. , Also, charged residues in the so-called coiled-coil region of the gamma domain are known to be involved in substrate HPG activation; , however, the exact mechanism whereby this region is important catalytically in substrate catalysis (namely, enhancing substrate turnover without conferring substrate affinity per se) has been elucidated only recently . Even though the above-mentioned studies do provide an identity to the sites whereby substrate HPG interacts with the SK·HPG complex, whether the sites act independently or in concert remains an important mechanistic question demanding a resolution.…”

Structural Biology and Protein Engineering of Thrombolytics

“…The domain β (residues 151–287) is responsible for high-affinity binding during the activation of the plasminogen “partner” [197,[204], [205], [206]] but it also facilitates the plasminogen “substrate” binding and processing [[207], [208], [209], [210], [211]]. Finally, the domain γ (residues 288–414) is involved in stabilizing the streptokinase-plasminogen complex and in inducing its proteolytic activity [193,197,201,204,208,[212], [213], [214]]. Although having one or two major functions, each domain participates in all the steps of plasminogen activation due to the high level of cooperativity [206,215,216].…”

“…Finally, the Arg561-Val562 peptide bond of the “substrate” molecule is hydrolytically cleaved by the “partner’s” catalytic triad His603, Asp646, and Ser741, and the final molecule of active plasmin is formed and released [201,217,219]. Regions 88–97, 164–186, and 314–342 of streptokinase were reported to be important for the plasminogen substrate processing during this final step of activation [208,209,213,215,224]. When traces of plasmin molecules are generated, streptokinase tends to form the streptokinase-plasmin complex preferentially due to the approximately three orders of magnitude higher affinity towards plasmin compared to plasminogen.…”