The compact domain conformation of human Glu-plasminogen in solution

Streptokinase (SK) 1 is used clinically as a thrombolytic drug to activate plasminogen (Pg) to plasmin (Pm), the serine proteinase responsible for dissolution of fibrin clots (1). Native [Glu]Pg is a multidomain zymogen consisting of a 77-residue N-terminal peptide, five kringle domains, and a serine proteinase catalytic domain that is activated by cleavage of Arg 561 -Val 562 (2, 3).[Glu]Pg is in a compact conformation, maintained by intramolecular interactions between the N-terminal peptide and lysine-binding sites in kringles 4 and 5 (4 -6). Release of the N-terminal peptide by Pm cleavage generates [Lys]Pg, which adopts an extended conformation and is cleaved by plasminogen activators at a faster rate (5, 7-13). SK possesses no intrinsic catalytic activity but interacts with Pg and Pm, converting both the zymogen and active proteinase into specific proteolytic Pg activators (14 -19). SK binding to Pg results in conformational expression of an active catalytic site on the zymogen without the usual strict requirement for peptide bond cleavage (14,16,17). Pm is generated subsequently by proteolytic cleavage of Arg 561 -Val 562 , and the SK⅐Pm complex propagates Pg activation through expression of a substrate recognition exosite (20,21).It is well established that both conformational and proteolytic activation contribute to SK-induced Pg activation, but there are a number of unresolved questions concerning the mechanism of conformational activation and its coupling to subsequent proteolytic Pm formation. Early studies (14,16,17) demonstrated that interaction of SK with Pg produced the activated Pg catalytic site in the SK⅐Pg* complex. Subsequent kinetic studies indicated that Pg activation involved an initially formed SK⅐Pg* activation complex and an isomerized form of the complex (22, 23). The isomerization was time-, chloride ion-, and fibrinogen-dependent, and the active complexes interconverted slowly under certain experimental conditions. In other studies, a species of Pg isolated from a mixture of SK and Pg was reported to be an active "virgin enzyme" form of free Pg*, suggesting irreversible conformational activation (24). The relationship between SK-Pg binding and conformational activation and its reversibility have not been clearly established.Previous studies of the mechanism of Pg activation by SK have not taken into account the binding affinities of SK for Pg and Pm species, primarily because consistent and reliable equilibrium binding constants have not been available. A number of studies have examined the binding interactions by various experimental approaches but have resulted in a very broad range of dissociation constants, varying from 28 pM to 220 nM for [Glu]Pg (25-32), for example, where the higher affinities may result from Pm generation in SK/Pg mixtures. Our equilibrium binding studies have employed active site-labeled fluorescent Pg and Pm analogs to quantitate SK binding in the absence of proteolytic reactions (21,28,33,34). The results for the active site-labeled proteins support a rela...

show abstract

“…Under conditions where [Pg] free Ͻ Ͻ K S which were found to apply, Equation 4 can be reduced to Equation 10.…”

Section: Substitution Of Equation 3 Into Equation 2 Gave Equationmentioning

confidence: 99%

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

Boxrud¹,

Verhamme

Bock

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The integrity of Tyr 512 is required for reactivity of K5 with its ligands (41). The same residue is also essential for the interaction of K5 with Pg residue Lys 50 , which stabilizes Pg in a closed conformation when the protein is in the circulation (42).…”

Section: Analyses Of Sequence Similarities Between Sk and Humanmentioning

confidence: 99%

The Voltage-dependent Anion Channel Is a Receptor for Plasminogen Kringle 5 on Human Endothelial Cells

Gonzalez-Gronow

Kalfa

Johnson

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Human plasminogen contains structural domains that are termed kringles. Proteolytic cleavage of plasminogen yields kringles 1-3 or 4 and kringle 5 (K5), which regulate endothelial cell proliferation. The receptor for kringles 1-3 or 4 has been identified as cell surfaceassociated ATP synthase; however, the receptor for K5 is not known. Sequence homology exists between the plasminogen activator streptokinase and the human voltagedependent anion channel (VDAC); however, a functional relationship between these proteins has not been reported. A streptokinase binding site for K5 is located between residues Tyr 252 -Lys 283 , which is homologous to the primary sequence of VDAC residues Tyr 224 -Lys 255 . Antibodies against these sequences react with VDAC and detect this protein on the plasma membrane of human endothelial cells. K5 binds with high affinity (K d of 28 nM) to endothelial cells, and binding is inhibited by these antibodies. Purified VDAC binds to K5 but only when reconstituted into liposomes. K5 also interferes with mechanisms controlling the regulation of intracellular Ca 2؉ via its interaction with VDAC. K5 binding to endothelial cells also induces a decrease in intracellular pH and hyperpolarization of the mitochondrial membrane. These studies suggest that VDAC is a receptor for K5.

show abstract

“…NTP segment in determining the Pgn global fold (Castellino et al, 1973;Sjoholm et al, 1973;Claeys & Vermylen, 1974;Violand et al, 1975;Markus et al, 1978Markus et al, , 1979Tranqui et al, 1979;Binyai & Patthy, 1984;Mangel et al, 1990;Ramakrishnan et al, 1991 ;Ponting et al, 1992a;Marshall et al, 1994;Weisel et al, 1994). …”

Section: _mentioning

confidence: 99%

“…The interaction was found to be interfered by the presence of L-lysine or 6-AHA, a zwitterionic lysine analog. Thus, in intact Pgn, the NTP domain has long been suspected of interacting with lysinebinding kringle (K) modules, contiguous within the segment that upon activation becomes the Plm heavy chain (reviewed by Ponting et al, 1992b). Reportedly, the interaction could involve KI , K4 (Vali & Patthy, 1982;Ramesh et al, 1995), and/or K5 (Christensen, 1984).…”

mentioning

confidence: 99%

Structural/functional properties of the Glu1‐HSer57 N‐terminal fragment of human plasminogen: Conformational characterization and interaction with kringle domains

Martí

Carreño

et al. 1998

Protein Science

View full text Add to dashboard Cite

The Glul-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of -9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 'H-NMR at 500 MHz and values for the equilibrium association constants (K,) determined: rK1, K, -4.6 m"' ; rK2, K, -3.3 mM"; K4, KO -6.2 m"' ; K5, KO -2.3 mM". Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Ne-acetyl-L-lysine methyl ester ( K , < 0.6 mM"), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3, which is devoid of a LBS.CB-NTP and L-NTP 'H-NMR spectra were assigned and interproton distances estimated from 'H-'H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-AspGlu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys50 within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectraand exhibit fast amide 'H-2H exchange in 2H20, suggestive of high flexibility.Keywords: kringle domains; plasminogen activation peptide; plasminogen N-terminal peptide; preactivation peptide structure Reprint requests to: M. Llinas, Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213; e-mail: Ilinas+ @andrew.cmu.edu. Abbreviations; ID, one-dimensional; 2D, two-dimensional; 6-AHA, 6-aminohexanoic acid; AcArgOMe, N"-acetyl-L-arginine methyl ester; AcLysOMe, N"-acetyl-L-lysine methyl ester; Acm, acetamidomethyl group; ADC, anti-distance constraints; CB-NTP, CNBr cleaved N-terminal peptide from human plasminogen, Glul-HSer57; CD, circular dichroism; COSY, 2D chemical shift correlated spectroscopy; DIEA, N,N-diisopropylethylamine; DMF, N,Ndimethylformamide; DSS, 3-(trimethylsilyl)-l-propanesulfonic acid, sodium salt; EM, energy minimization; Et20, anhydrous diethyl ether; FmoclrBu, 9-fluorenylmethyloxycarbonyl/terf-butyl; HATU, N-[(dimethylamino) (lH-1,2,3-triazolo[4,5-b]pyridin-I-yl) methylenel-N-methyl methanaminium hexafluorophosphate N-oxide; HOAt, 7-aza-I-hydroxy-benzotriazole; HOAc, glacial a...

show abstract

The compact domain conformation of human Glu-plasminogen in solution

Cited by 46 publications

References 45 publications

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

The Voltage-dependent Anion Channel Is a Receptor for Plasminogen Kringle 5 on Human Endothelial Cells

Structural/functional properties of the Glu1‐HSer57 N‐terminal fragment of human plasminogen: Conformational characterization and interaction with kringle domains

Contact Info

Product

Resources

About