Crystal structure of the complex between thrombin and the central “E” region of fibrin

A family of serine proteases (SPs) mediates the proteolytic cascades of embryonic development and immune response in invertebrates. These proteases, called easter-type SPs, consist of clip and chymotrypsin-like SP domains. The SP domain of easter-type proteases differs from those of typical SPs in its primary structure. Herein, we report the first crystal structure of the SP domain of easter-type proteases, presented as that of prophenoloxidase activating factor (PPAF)-I in zymogen form. This structure reveals several important structural features including a bound calcium ion, an additional loop with a unique disulfide linkage, a canyon-like deep active site, and an exposed activation loop. We subsequently show the role of the bound calcium and the proteolytic susceptibility of the activation loop, which occurs in a clip domain-independent manner. Based on biochemical study in the presence of heparin, we suggest that PPAF-III, highly homologous to PPAF-I, contains a surface patch that is responsible for enhancing the catalytic activity through interaction with a nonsubstrate region of a target protein. These results provide insights into an activation mechanism of easter-type proteases in proteolytic cascades, in comparison with the well studied blood coagulation enzymes in mammals.

Section: Discussionmentioning

confidence: 99%

Crystal Structure of the Serine Protease Domain of Prophenoloxidase Activating Factor-I

Piao

Kim

et al. 2007

“…The exosite both confers substrate specificity so that only selected bonds are cleaved but can also change substrate specificity as demonstrated by the thrombinthrombomodulin complex (62). Thrombin and pCDK2/cyclin A are similar in that in their tight inhibitor complexes (thrombinhirudin or pCDK2/cyclin A-p27) there is a direct stereochemical path between the catalytic site and remote site, whereas in substrate complexes (thrombin/E region of fibrin (63) or pCDK2/cyclin A-CDC6 peptide) a path has been modeled but not observed directly. The studies with pCDK2/cyclin A and the substrate recruitment peptides and bispeptides have been informative and shown the power of the recruitment site for a potent inhibitor.…”

Section: Discussionmentioning

confidence: 99%

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

Cheng

Noble

Skamnaki

et al. 2006

Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 Å from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the ␥-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower K m compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/ cyclin A activity against a heptapeptide substrate (K i ‫؍‬ 0.83 nM) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (K D 0.4 M) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.

“…Crystallographic studies have revealed the nature of individual interactions at exosites 1 and 2 with fibrinogen fragment E and the ␥Ј-peptide, respectively (40,41). When thrombin is bound to ␥ A /␥Ј-fibrin, both of its exosites are ligated simultaneously.…”

Section: Discussionmentioning

confidence: 99%

Bivalent Binding to γA/γ′-Fibrin Engages Both Exosites of Thrombin and Protects It from Inhibition by the Antithrombin-Heparin Complex

Fredenburgh

Stafford

Leslie

et al. 2008

109

Thrombin exosite 1 binds the predominant ␥ A /␥ A -fibrin form with low affinity. A subpopulation of fibrin molecules, ␥ A /␥-fibrin, has an extended COOH terminus ␥-chain that binds exosite 2 of thrombin. Bivalent binding to ␥ A /␥-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from ␥ A /␥-fibrin clots than from ␥ A /␥ A -fibrin clots. After 24 h of washing, however, both ␥ A /␥-and ␥ A /␥ A -fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparinthrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in ␥ A /␥-fibrin clots than they were in ␥ A /␥ A -fibrin clots. This difference reflects bivalent binding of thrombin to ␥ A /␥-fibrin because (a) it is abolished by addition of a ␥-chaindirected antibody that blocks exosite 2-mediated binding of thrombin to the ␥-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with ␥ A /␥-fibrin than with ␥ A /␥ A -fibrin clots. Thus, bivalent binding of thrombin to ␥ A /␥-fibrin protects thrombin from inhibition, raising the possibility that ␥ A /␥-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.