3-Nitrotyrosine (NT) is ;103 -fold more acidic than Tyr, and its absorption properties are strongly pH-dependent. NT absorbs radiation in the wavelength range where Tyr and Trp emit fluorescence (300-450 nm), and it is essentially nonfluorescent. Therefore, NT may function as an energy acceptor in resonance energy transfer (FRET) studies for investigating ligand-protein interactions. Here, the potentialities of NT were tested on the hirudin-thrombin system, a well-characterized protease-inhibitor pair of key pharmacological importance. We synthesized two analogs of the N-terminal domain (residues 1-47) of hirudin: Y3NT, in which Tyr3 was replaced by NT, and S2R/Y3NT, containing the substitutions Ser2 ! Arg and Tyr3 ! NT. The binding of these analogs to thrombin was investigated at pH 8 by FRET and UV/Vis-absorption spectroscopy. Upon hirudin binding, the fluorescence of thrombin was reduced by ;50%, due to the energy transfer occurring between the Trp residues of the enzyme (i.e., the donors) and the single NT of the inhibitor (i.e., the acceptor). The changes in the absorption spectra of the enzyme-inhibitor complex indicate that the phenate moiety of NT in the free state becomes protonated to phenol in the thrombin-bound form. Our results indicate that the incorporation of NT can be effectively used to detect protein-protein interactions with sensitivity in the low nanomolar range, to uncover subtle structural features at the ligand-protein interface, and to obtain reliable K d values for structure-activity relationship studies. Furthermore, advances in chemical and genetic methods, useful for incorporating noncoded amino acids into proteins, highlight the broad applicability of NT in biotechnology and pharmacological screening.

In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by approximately 2 kcal/mol (1 cal identical with 4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the 'fast' form. Finally, we have used hirudin fragment 1-47 as a molecular probe of the conformation of thrombin recognition sites in the fast and 'slow' form. From the effects of amino acid substitutions on the affinity of fragment 1-47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.

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Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active ␣-thrombin (␣T). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320 -Ile-321 to yield mature ␣T. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nM to 2 M), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active Pre2, a nicked Pre2 derivative with a single cleaved Ala-470 -Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470 -Asn-471 is instrumental for the onset of catalysis in Pre2, which was, however, reduced about 100 -200-fold compared with ␣T. Of note, Pre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.Blood coagulation is a finely regulated physiological process that culminates with the factor Xa-mediated conversion of the prothrombin (ProT) 3 zymogen to the active ␣-thrombin (␣T) enzyme, which in turn is responsible for the generation of insoluble fibrin and activation of platelets via the GpIb␣-PAR1 pathway (1, 2). ProT (ϳ72 kDa) is a vitamin K-dependent glycoprotein produced in the liver and circulating at a relatively high plasma concentration (0.1 mg/ml) (3). The domain architecture of ProT includes a Gla domain (residues 1-46), a kringle-1 (residues 65-143) and a kringle-2 (residues 170 -248) domain, and a chymotrypsin-like protease domain (residues 285-579) connected by three intervening linker regions (Lnk-1, -2, and -3) (4). Isolated factor Xa (FXa) has low intrinsic ProTconverting activity, but when it is assembled in the presence of Ca 2ϩ with cofactor Va in the prothrombinase complex on the platelet surface, its ability to activate ProT is increased by about 5 orders of magnitude (5). FXa cleaves ProT in a concerted manner at two sites, i.e. Arg-271 and Arg-320, but the order of peptide bond cleavage is highly context-dependent. On the platelet surface, FXa first cleaves ProT at Arg-271 generating the inactive precursor prethrombin-2 (Pre2), which is attacked by FXa at Arg-320 to generate the active ␣T species, formed by the polypeptide chains Thr-272-Arg-320 and Ile-321-Glu-579 (6). ...