Role of the 88–97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis

Yadav, Suman; Datt, Manish; Singh, Balvinder; Sahni, Girish

doi:10.1016/j.bbapap.2008.05.013

Cited by 17 publications

(26 citation statements)

References 36 publications

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“…This is achieved through the formation of a substrate recognition exosite in the activator complex which mediates the specific recognition of substrate plasminogen for proteolytic activation [123]. The α domain of streptokinase contributes to the formation of this exosite and is critical for the activator complex to capture, extend and process substrate Glu-plasminogen [112,124]. Specifically inhibiting streptokinase α domain interactions would therefore hinder streptokinase mediated plasminogen activation through numerous mechanisms.…”

Section: Novel Therapeutics For the Treatment Of Invasive S Pyogenesmentioning

confidence: 99%

The Role of Streptokinase as a Virulence Determinant of Streptococcus pyogenes – Potential for Therapeutic Targeting

McArthur

Cook²,

Venturini³

et al. 2012

CDT

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. (2012). The role of streptokinase as a virulence determinant of streptococcus pyogenes -potential for therapeutic targeting. Current Drug Targets, 13 (3), 297-307. The role of streptokinase as a virulence determinant of streptococcus pyogenes -potential for therapeutic targeting AbstractStreptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome. These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein. Here, we describe the role of streptokinase in invasive pathogenesis and discuss some potentially useful strategies for disruption of streptokinase mediated plasminogen activation which could be employed to treat severe invasive S. pyogenes infections. AbstractStreptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome.These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein.Here, we describe the role of streptokinase in invasive pathogenesis and discuss some potentially useful strategies that disrupt streptokinase mediated plasminogen activation and could be employed to treat severe invasive S. pyogenes infections.3

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Section: Novel Therapeutics For the Treatment Of Invasive S Pyogenesmentioning

confidence: 99%

The Role of Streptokinase as a Virulence Determinant of Streptococcus pyogenes – Potential for Therapeutic Targeting

McArthur

Cook²,

Venturini³

et al. 2012

CDT

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“…Each of the three domains of SK has been implicated in this regard (29,30,35,36), and binding of two Pg molecules to the residue 1-59 sequence of the ␣-domain has been reported (36). In particular, segments 16 -36, 41-48, 48 -59, and 88 -97 of the SK ␣-domain have been concluded to play a role in Pg substrate recognition (32,33,37,38). For several SK mutants, a complex mixture of functional effects on their binding to [Glu]Pg and its conformational and proteolytic activation has been reported (28,31,33).…”

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confidence: 99%

Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase β-Domain and Kringle 5 of the Substrate

Tharp

Laha

Panizzi

et al. 2009

Journal of Biological Chemistry

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“…Moreover, the observation that the ␥ domain binds closely to the catalytic active center of the SK⅐HPN complex suggests that it might indeed be a preferred area of substrate interaction also (17), and there is, therefore, a need to explore intensively whether this domain contains any site(s) playing a role specifically in substrate interaction under conditions that unambiguously rule out the involvement of any 1:1 interactions. Previously, alanine mutagenesis of the extensive charged side chains of the residues present in the coiled coil region of the ␥ domain of SK has revealed the overall functional relevance of this region in HPG activation in general (29), but to look for the possible presence of a discrete substrate-interacting exosite/epitope of the type demonstrated in the ␣ and ␤ domains (20,21,35,41), in the present study we took recourse to a random mutagenesis approach.The results, presented below, examine a series of randommutant clones of the C-terminal region of SK (residues 210 -414) for their functional importance in substrate HPG activation. Because this approach initially yielded several scores of functionally compromised mutants that carried multiple-site mutations, an analysis of their "deconvolved" forms, that is, all or most single-site mutants derived from the mutant-clusters with compromised activity, was then carried out to decipher the functional importance of residues present in the ␥ domain in substrate recognition and/or turnover.…”

mentioning

confidence: 99%

“…Mechanistic insights on the specificity switch engendered by SK upon binding onto the HPN active site are crucial in major part because of possibly enabling the engineering of altogether new target specificities into pre-existent protease active sites for biomedical applications (20,21). The binding of SK to HPN results in the expression of substrate recognition "exosites" on the SK⅐HPG/HPN activator complex, which are located physically away from the active center of the enzyme (19).…”

mentioning

confidence: 99%

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

Yadav¹,

Aneja²,

Kumar³

et al. 2011

Journal of Biological Chemistry

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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...

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Role of the 88–97 loop in plasminogen activation by streptokinase probed through site-specific mutagenesis

Cited by 17 publications

References 36 publications

The Role of Streptokinase as a Virulence Determinant of Streptococcus pyogenes – Potential for Therapeutic Targeting

The Role of Streptokinase as a Virulence Determinant of Streptococcus pyogenes – Potential for Therapeutic Targeting

Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase β-Domain and Kringle 5 of the Substrate

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

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