Complexation of the Tissue Plasminogen Activator Protease with Benzamidine-type Inhibitors:  Interference by the Kringle 2 Module

Abstract

Well-resolved high-field 1H NMR signals between -0.1 and -0.7 ppm afford convenient probes to monitor the conformational state of the tissue plasminogen activator (tPA) protease, modulated by covalent inhibitor binding or activation cleavage [Hu, C.-K., Kohnert, U., Wilhelm, O., Fischer, S., & Llinas, M. (1994) Biochemistry 33, 11760-11766]. We have investigated recombinant BM 06.022 (a domain-deletion variant mutant from Escherichia coli comprising the kringle 2 and protease modules) and protease constructs of tPA in both single-chain (sc) and two-chain (tc) forms. The two proteins were studied when confronted with the noncovalent (i.e., reversible) active site inhibitors benzamidine and a series of bisbenzamidine derivatives: 2,5-bis(4-amidinobenzylidene)cyclopentanone, 2,6-bis(4-amidinobenzylidene)cyclohexanone, 2,7-bis(4-amidinobenzylidene)cycloheptanone, and 2,8-bis(4-amidino- benzylidene)cyclooctanone. At pH* 4.6, the 1H NMR spectrum is sensitive to complexation of the protease module with the various effectors. The amplitude of the inhibitor-shifted resonances is more pronounced for the tc-protease than for the sc-protease, suggesting that access of inhibitors to the protease catalytic site is facilitated upon conversion to the tc form. The effects detected by the NMR spectrum suggest a biphasic process, involving stronger (primary) and weaker (secondary) bindings to a single protease active site. Binding to the protease module in tc-BM 06.022 essentially generates the same spectral characteristics as detected upon binding to the isolated tc-protease construct. In contrast, a negligible perturbation by the inhibitors is observed on the (sc) BM 06.022. Hence, in the intact BM 06.022 the kringle 2 is structurally coupled to the protease module thus interfering with inhibitor molecules from accessing the protease active site. These domain-domain interactions relax upon conversion to the catalytically active tc form, thus decoupling the kringle 2 from the protease module in BM 06.022 while simultaneously exposing the active site to become accessible to effectors or substrates.

The effects of N‐ethyl‐N′‐methyl imidazolium chloride on the solubility, stability and aggregation of tc‐rPA

“…Unfortunately, rPA cleaved itself slowly to its more active two‐chain form (tc‐rPA) upon incubation at low salt (EMIMCl) concentrations. Cleavage took place between Arg275 and Ile276 leaving the two protein domains only covalently connected through a disulfide bridge between Cys264 und Cys393 . This was confirmed via RP‐HPLC and reducing SDS/PAGE (Fig.…”

“…S1. rPA has very low solubility under physiological conditions and effective refolding to the native state requires both the presence of a redox shuffling system and cosolvent additives which enhance the solubility of the native and the unfolded states . Here, we examine the physical chemistry of how various concentrations of EMIMCl affect the efficiency of protein refolding.…”

The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1

“…Binding of tPA and substrate plasminogen to promoter templates such as fibrin plays a major role in stimulating and regulating enzyme activity in fibrinolysis. More subtle intramolecular interactions between tPA binding domains and the serine protease domain have also been identified by calorimetry between the finger and/or growth factor domain and the protease domain [22]; and by NMR observations of binding between the kringle 2 and protease domains [23]. Hence fibrin binding by both finger and kringle domains may affect the protease domain and these interdomain interactions may be different in sc‐ and tctPA [23].…”

Ferrocene‐Donor and 4,5‐Dicyanoimidazole‐Acceptor Moieties in Charge‐Transfer Chromophores with π Linkers Tailored for Second‐Order Nonlinear Optics

“…in 1992. This protein was reported to have a low solubility above pH 4.6 and is mostly insoluble in the pH range of 5 to 8 29. Besides native rPA, we included fully S ‐carboxymethylated rPA (IAA‐rPA) in the solubility studies as a model for non‐native, denatured protein states.…”