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

Abstract

The selective deletion of a discrete surface-exposed epitope (residues 254–262; 250-loop) in the β domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SKdel254–262). A kinetic analysis of SKdel254–262 revealed that its low HPG activator activity arose from a 5–6-fold increase in Kmfor HPG as substrate, with little alteration in kcatrates. This increase in the Kmfor 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 SKdel254–262. The involvement of kringles was further indicated by a hypersusceptibility of the SKdel254–262· plasmin activator complex to ε-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 SKdel254–262·HPG was reduced by about 3-fold in comparison with that of nSK·HPG. Overall, these observations identify the 250 loop in the β 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. The selective deletion of a discrete surface-exposed epitope (residues 254–262; 250-loop) in the β domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SKdel254–262). A kinetic analysis of SKdel254–262 revealed that its low HPG activator activity arose from a 5–6-fold increase in Kmfor HPG as substrate, with little alteration in kcatrates. This increase in the Kmfor 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 SKdel254–262. The involvement of kringles was further indicated by a hypersusceptibility of the SKdel254–262· plasmin activator complex to ε-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 SKdel254–262·HPG was reduced by about 3-fold in comparison with that of nSK·HPG. Overall, these observations identify the 250 loop in the β 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. streptokinase plasminogen (irrespective of source) human plasminogen human plasmin microplasmin microplasminogen kringles 1–5 of plasminogen p-nitrophenyl p-guanidinobenzoate splicing-overlap-extension ε-amino caproic acid native-like SK Streptokinase (SK),1 a bacterial protein secreted by the Lancefield Group C β-hemolytic streptococci, is widely used as a thrombolytic agent in the treatment of various circulatory disorders, including myocardial infarction (1.International Study of Infarct Survival-3Lancet. 1992; 339: 753-781Abstract PubMed Scopus (1145) Google Scholar). Unlike other human plasminogen (HPG) activators, like tissue plasminogen activator and urokinase, SK does not possess any intrinsic enzymatic activity. Instead, SK forms an equimolar, stoichiometric complex with “partner” HPG or plasmin (HPN), which then catalytically activates free “substrate” molecules of HPG to HPN by selective cleavage of the Arg561-Val562 peptide bond (2.Castellino F.J. Chem. Rev. 1981; 81: 431-446Crossref Scopus (122) Google Scholar, 3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar). It is believed that consequent to the initial SK·HPG complexation, there is a structural rearrangement within the complex, and even before any proteolytic cleavage takes place, an active center within the HPG moiety capable of undergoing acylation is formed (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar). This activated complex is rapidly transformed into an SK·HPN complex and develops an HPG activator activity. Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK·HPN displays a very narrow substrate specificity (4.Markus G. Werkheiser W.C. J. Biol. Chem. 1964; 239: 2637-2643Abstract Full Text PDF PubMed Google Scholar). 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 the of active with and J. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). SK to of similar and by and the and J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The of the domain of HPN complexed with SK SK the substrate specificity of HPN by a or in which the macromolecular substrate the scissile peptide bond for cleavage by the HPN active a narrow substrate onto an active In SK does not to any in the active with to a on which the substrate in the of the HPG activation loop the active center of the complex Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). the of these and to the of the a the in substrate (4.Markus G. Werkheiser W.C. J. Biol. Chem. 1964; 239: 2637-2643Abstract Full Text PDF PubMed Google the binding of SK to HPN in a of the for peptide little alteration in as with free HPN, that the specificity of the active center of HPN SK binding in for even the peptide the alteration of the macromolecular substrate specificity of HPN by SK is to to on the SK·HPN complex, as by the of active HPN J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies in indicated that on the of SK, from in the and β inhibition for HPG activation by the preformed SK·HPN complex under conditions where the of SK and HPN was essentially G. PubMed Scopus Google Scholar, G. PubMed Scopus Google the of SK complexed with J. PubMed Scopus Google a of of the in the complexation, the between the activator complex and substrate This is to the binary of the complex an of a substrate in the β and a in and substrate of the kringles that to important in HPG PubMed Scopus Google Scholar). a and of the of in the binary complex, discrete of SK, that in the by the activator complex not in the active center PubMed Scopus Google and in SK in the β domain G. PubMed Scopus Google Scholar, PubMed Scopus Google to important for HPG activation a of a structural in substrate HPG affinity onto the activator complex not the of SK, the β domain displays affinity for HPG J. PubMed Scopus Google the domain displays affinity for HPG with the domain affinity of for and G. and structural studies that and β in the substrate J. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). studies in the β domain to important to that these in substrate by the binary complex PubMed Scopus Google Scholar). of the of the free β domain J. PubMed Scopus Google and its comparison with the other two SK similar not revealed the of a loop in the β domain 250-loop) that into the In the to loop on the that structural is in substrate discrete deletion of loop to a selective increase in of the activator complex. The of the by loop in substrate and identify a important of the macromolecular substrate-specific in the SK·HPN complex, which via the kringle(s) in was from or from human by affinity PubMed Scopus Google Scholar). The and of was from and and other from by HPN was by with on a of HPG in and for J. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). other of the A of and used in to splicing-overlap-extension PubMed Scopus Google in which in the G. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). for for the of SKdel254–262 as The as The as for for the of and as of the as of the as proteins in with essentially to the of the The for the of the to SK from of an as to the protein as G. PubMed Scopus Google Scholar). protein analysis of the SK in to as nSK was to its a The was in the mutant from the The and the proteins in the with G. PubMed Scopus Google Scholar). This in was then on a onto a a of in The proteins as by proteins in as The was in and under conditions for to a the protein in the was by with The β domain was then to by on a in the domain of plasminogen (residues of kringles was by cleavage of HPG by HPN under conditions was from the by a by a to HPN and as J. Biol. Chem. Full Text PDF PubMed Google Scholar). The was then to by on a of The of formed was by which showed a the from its J. Biol. Chem. Full Text PDF PubMed Google Scholar). The proteolytic of the HPG was by HPG with HPN of HPG and under conditions for these the proteolytic conversion of HPG to was to with HPN was from the by a This was by on to and The of was by analysis J. Biol. Chem. Full Text PDF PubMed Google Scholar). with urokinase, which is to a activator of of the of J. Biol. Chem. Full Text PDF PubMed Google was used to that the activation of used as substrate, was with that SK·HPN as the activator from and to a and The in was to the of activation G. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). HPG was to an assay and and the of was as a of (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, PubMed Scopus Google Scholar). of HPG to the assay of and substrate and the in was as a of the of HPG activation by by of preformed to the assay of substrate HPG J. Biol. Chem. Full Text PDF PubMed Google Scholar). the the of HPN active was the (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). and HPN in for in and an of the was to a assay and of the substrate to a of complex in the The was for The kinetic by PubMed Scopus Google Scholar). and between HPG and the to as binary in by resonant mirror-based the J. Scopus Google Scholar, Scopus Google Scholar). In these was on to the This was by the of HPG to the on the HPG was then by with by with The for the HPG onto the was in in and The was in to the with binding of nSK or SKdel254–262 and binding was during the the was with binding and the was PubMed Scopus Google Scholar). of the was by with and was with binding In in the in the was as a for the binding In experiments where was used to its on the binary complex was formed between and HPG in binding The of the binary was by the with of The of the and the kinetic from the by of the and to the by the the of to the to the observed the of was to the from the of the Biochem. PubMed Scopus Google Scholar). The was from the of of The was then as of in to by analysis of the of not mirror was used to the and between or and with a substrate binding to binary complex and to as ternary In binary interaction it was that was to a and SK·HPG binary complex The of complex is very to the of the SK·HPG complex, as observed by J. PubMed Scopus Google Scholar). the complex to the which even with It that SK was with was the binary complex it was binary complex SK and to HPG PubMed Scopus Google Scholar). to of the SK·HPG binary complex by to ternary substrate interaction under conditions that not the of the binary In contrast, was to to ternary complex In a ternary interaction a binary complex was formed by a of nSK or SKdel254–262 onto on on the binary complex with binding and with a was of HPG or then to the binding by the the was with binding and the was of the was by the ternary with by with binding It was that the interaction of ternary HPG with the binary complex, the binary complex The was to a to and of the kinetic for ternary complex and In to was experiments in which the ternary complex and experiments various a was to binary complex by and as It was observed that the not significantly a of as a of In experiments where was used as ternary was to to the with binding in of and and as and the as The of on the binding of ternary HPG to complexed with HPG was by the binding in a of substrate HPG in the of the analysis of these the binding in was as and of of ternary as a of to binding for nSK and SKdel254–262. the of was for of preformed between HPG and nSK or SKdel254–262 in the of substrate of proteins in on a from to 250 in a and the was from the protein The was an of F.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the domain of HPG as a of HPG and of SK β domain and the complex from the and The β domain in including the J. PubMed Scopus Google Scholar). The in complex with of the β domain J. PubMed Scopus Google Scholar). The β domain was on the β domain of the complex, and the of was The β domain the it is for docking for a HPG domain was the as a with the of PubMed Scopus Google with and the of was then onto the two of on the The of kringles is to with the loop of the SK and these of in the SK complex. of the is the of the This was not for The other was then into of the of SK β The complex is in the of the showed to the of the complex. The of docking revealed that the C of the domain is within of the of the as in the the of and the the two and the between peptide that the onto in the in the of the its involvement in the of substrate specificity of HPN by a deletion mutant the PubMed Scopus Google Scholar). The deletion was by of of the for other This was by of the in a PubMed Scopus Google Scholar). the that a of the β the two the loop and a of between the J. PubMed Scopus Google Scholar). The the in to and the of the protein of was then by the of SKdel254–262 was then from the to a G. PubMed Scopus Google Scholar). SKdel254–262 was for its to substrate it showed a specific activity that was about that of native-like SK and the and the deletion of the in in the of SK as by its by The of nSK and the mutant however, not any not a native-like of the mutant in and the of the deletion in the β domain that in the of the from of the two of SK in the a deletion the in a for the β The for the β domain and then in and for and no observed in the of with to in not that the of SK that of the β domain by the deletion of the loop from the the deletion of the in any alteration in the affinity of the mutant with the of interaction between HPG and nSK with of SKdel254–262 by the resonant mirror a for in the of on the of kinetic experiments by nSK or SKdel254–262 onto an by the The of and of HPG with then as under In HPG was on on a and the binding with SK as these between and observed for the binding of which is with a of the for the interaction to a a was observed between and to the The that the mutant an affinity that was not significantly from that of nSK and and for the binding of HPG to the of HPG was on onto a of the then as under The was the The of was to the from the of The was from the of of The was from the of kinetic as in a new HPG was on onto a of the then as under The was the The of was to the from the of The was from the of of The was from the of kinetic as experiments by on as by other J. PubMed Scopus Google F.J. J. PubMed Scopus Google similar binding for nSK and SKdel254–262 not that the does not significantly in the binding interaction between SK and further the for of activator activity of the loop deletion like the it the active in the active to the that SK and HPG (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar, PubMed Scopus Google was observed with the SK HPG the once the two in is with a observed to the of a SK·HPG complex and consequent acylation the active of HPG with (3.McClintock D.K. Bell P.H. Biochem. Biophys. Res. Commun. 1971; 43: 694-702Crossref PubMed Scopus (153) Google Scholar). the nSK as a was with in the of the was A similar was in the of SKdel254–262 as the of these was of that observed with SK from not It that a free PubMed Scopus Google and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google for the of HPG by the of that of SK, was analysis of the nSK and SKdel254–262 showed that of the protein a free the to by P.H. G. PubMed Scopus Google Scholar). The mutant was then further for its to HPG by of its with In it a of of activity with to nSK that it the active in these that the observed HPG activator activity of SKdel254–262 was not to any on its to HPG the binary complex a of the of the active the kinetic with the substrate binding and of macromolecular substrate (HPG) and the low peptide substrate by the of HPN with nSK or SKdel254–262. the for the kinetic analysis of the activation of substrate HPG by the complex revealed a 5–6-fold increase in the for HPG with that of with little alteration in the This indicated that the mutant formed an activator complex with HPN that an decreased affinity for the macromolecular substrate as with that of the the other the by or for the peptide substrate essentially as with that of HPN that the specificity of the active in HPN the for substrate was decreased as with that of kinetic for HPG activation by of HPN and kinetic for substrate HPG activation as under The the of similar for the activation of substrate HPG by SKdel254–262 not in a new The kinetic for substrate HPG activation as under The the of similar for the activation of substrate HPG by SKdel254–262 not It is that the of SK with free HPN to an reduced of the HPN active by and to about by SK binding in the of the active is for in a increase in the for the of by SK·HPN complex with that of HPN J. PubMed Scopus Google Scholar, 1981; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The increase in of HPN for observed with the β domain G. PubMed Scopus Google Scholar). The on the of the in SKdel254–262 that the with a in that is to the active a that is with its in substrate HPG to the activator complex and with peptide inhibition experiments G. PubMed Scopus Google Scholar). the for the of the low peptide substrate by the HPN complex of the deletion mutant demonstrate that the affinity for macromolecular substrate (HPG) was decreased in the of a alteration in the of the low peptide kinetic for activity of of HPN and the of the and HPN in an and an of was for of as under The the of in a new the of the and HPN in an and an of was for of as under The the of The on for HPG to the affinity of substrate HPG with activator between HPG and on the and HPG with SKdel254–262 on the a a new assay for ternary complex between SK and two molecules of in the binary and the other as a substrate, was on a resonant mirror-based a analysis of the of a ternary complex with substrate HPG onto the binary complex of HPG and nSK on J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to the of ternary complex with substrate HPG onto the preformed binary SK·HPG complex on resonant mirror used two of the ternary complex, and of the preformed binary complex, to and of binary and ternary to HPG binding for It that the preformed SK·HPG complex is highly to the of the activator complex on the substrate is to inhibition in the low PubMed Scopus Google a that the of the interaction of substrate HPG with preformed SK·HPG complex. This of the preformed binary complex and of the ternary complex to to the interaction of ternary HPG with the SK·HPG binary complex. to the low affinity of the substrate HPG the SK·HPG binary complex J. Biol. Chem. Full Text PDF PubMed Google a of substrate HPG was the binary to its the and low of and and and for the interaction of substrate and with complexed with for the of substrate and with nSK HPG or SKdel254–262 HPG binary complex by the to the binding as under A binary complex between and HPG onto the was and then the binding of of substrate HPG or was in a new for the of substrate and with nSK HPG or SKdel254–262 HPG binary complex by the to the binding as under A binary complex between and HPG onto the was and then the binding of of substrate HPG or was interaction studies of HPG with SK·HPG binary complex assay J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an assay a of affinity in of it to into the of the interaction of the activator complex with the macromolecular The for the interaction of substrate and with nSK or SKdel254–262 complexed with HPG in It is that substrate HPG with the SKdel254–262·HPG binary complex with a affinity as with binary complex This decrease in affinity is to the increase in the for substrate HPG enzymatic activity as the for between nSK and the of the in substrate HPG molecules by the activator complex. studies that an substrate HPG of the specificity of the HPN active the of in the macromolecular substrate specificity of HPN J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). The structural basis an the in substrate specificity of the HPN active consequent to SK binding however, essentially The in the of the β domain as an important determinant of the macromolecular substrate specificity of the SK·HPG activator complex. The of two the of the that with the in substrate HPG the the of kringles in substrate the of nSK and the mutant in the of of a that is to HPG activation by SK as as ternary complex a (2.Castellino F.J. Chem. Rev. 1981; 81: 431-446Crossref Scopus (122) Google Scholar, PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies to the of of on substrate HPG activation by and showed that the mutant a to inhibition of substrate HPG activation nSK in a that not the activity of the two binary or the activity of free It PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a binding that the docking of substrate HPG onto SK·HPG binary complex formed on a does not the of the rates of binding and of substrate HPG with the affinity binary complex. to the of on the ternary of substrate HPG to with on resonant mirror as SKdel254–262·HPG binary complex, an of was which is the with binary complex the that the binary complex of the with the with substrate in a that is as with to with the it is that of the between activator and substrate by the deletion of the the mutant to inhibition by The that the mutant is to of that other of the by the selective deletion of the the observed difference in substrate between and SKdel254–262·HPG activator is it is to that the rates of activation of substrate which is of HPG by nSK and SKdel254–262 not from was to the is to a substrate for the preformed activator complex J. Biol. Chem. Full Text PDF PubMed Google Scholar). kinetic studies as the substrate showed no difference between nSK and SKdel254–262 with to initial of substrate activation under conditions where the activator of the two and showed a difference the substrate the observations that binding in the in the of of the macromolecular substrate-specific in the activator complex. further that kringles in ternary binding experiments on the resonant where the binding of the and kringles 1–5 of to the binary complex preformed between nSK or the mutant with in with kinetic showed the for nSK and with was used as the docking substrate during ternary complex the binary complex showed an affinity of for that of the SKdel254–262·HPG complex was to a 3-fold affinity for nSK these that the of the β domain of SK with substrate HPG via the studies the between the β domain of SK and the was used as a of the HPG kringles and does not any in the complex however, for for that a the in a or any other is in interaction during the of of was that the involvement of in the binding with the β domain of SK F.J. J. PubMed Scopus Google Scholar). a that interaction the of binary and ternary complex is the loop its binding specificity or substrate on the in the a that is by a G. PubMed Scopus Google that a peptide the by SK·HPG binding and HPG activation by preformed SK·HPN activator complex. the other the does not the that the kringles in substrate and HPG to in the of the involvement of a discrete epitope in SK in substrate and binding via the kringle(s) of further studies to identify the of the kringle(s) the domain of the substrate the and in the formed by the activator complex. This in a of interaction the It however, that the even the of the the HPG activation with that loop is not the determinant of the SK·HPN studies that the domain as as the β to the of HPG specificity in the activator complex G. PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). The of the domain to with substrate HPG is from the J. PubMed Scopus Google it does by with kringle(s) of substrate is It is from that the β domain a affinity for HPG as G. PubMed Scopus Google Scholar). the that of in the β domain in in the for activator activity with little in the for HPG G. PubMed Scopus Google that other the in domain in the of the substrate specificity of the activator complex. observed and G. that of the to further increase in the for substrate HPG about that of with alteration in the of its complex with It is that the β domain a of the affinity of SK for HPG for the of the activator complex J. PubMed Scopus Google and of the affinity for the interaction of the with substrate as the This as to domain by possess HPG activator a two for a HPG activator protein to with and to then with in This a bacterial HPG is to as a in a to that of SK Biol. PubMed Scopus Google and of these HPG The ternary of that not to the active of the new that the substrate specificity of Scholar). of in in and SK in that in of substrate specificity the of HPG for the and and for to for the for the of SK was with the of and to of for the of