Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Abstract

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-A resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin beta-turn of the second inner loop pivots at Val64 and Asp70 by 60 degrees. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 beta-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle.

1H‐NMR assignments and secondary structure of human plasminogen kringle 1

“…In PgdK1, NOES are observed between Trp25 and Tyr50, consistent with the two peptide strands crossing over each other although without proper P-sheet formation. Finally, in contrast to the case for tPA/K2 [33, 361, uPA/K [37, 671, and Ptb/K2 [30], we have uncovered no evidence for a-helical segments in Pgn/Kl. Neither has an a-helix been observed in the crystallographic structures of b-Ptb/Kl [29] and PgdK4 [31] nor in the solution structures of Pgn/K4 [34, 351.…”

Binding of Exosite Ligands to Human Thrombin

“…In addition, Fbg bound to [ANS]FPRmeizothrombin-des-F1 with a K D value of 25 M and was competitively displaced by Hir 54 -65 (SO 3 Ϫ ), which bound with a K D value of 27 nM, in agreement with independent binding studies. 2 These results were in keeping with crystallographic studies of the thrombin-F2 complex (14,61) and meizothrombindes-F1 (56), which concluded that F2 does not cover exosite I or interfere directly with the active site. Meizothrombin-des-F1, which contains F2 covalently attached, has activity toward tripeptide chromogenic substrates, but little activity toward clotting of Fbg (62).…”

“…Exosite I binds fibrinogen (Fbg) 1 (3), fibrin I and II (3,4), the 12-residue carboxyl-terminal hirudin 54 -65 sequence (5,6), thrombomodulin (7), the thrombin receptor (8,9), and an acidic sequence on the serpin, heparin cofactor II (10,11). Exosite II binds heparin and other glycosaminoglycans (2,12,13), prothrombin activation fragment 2 (F2) (14), the chondroitin sulfate moiety of thrombomodulin (15,16), the leech peptide hemadin (17), and an exosite II-specific human monoclonal antibody (18). Factors V (19 -22), Va (21,22), and VIII (19), platelet glycoprotein Ib␣ (23)(24)(25), and the snake venom protein bothrojaracin (26) have been reported to interact with both exosites I and II.…”

The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin

“…One of these cationic exosites is the fibrinogen recognition site (Fenton, 1981(Fenton, , 1986, whereas the other is most likely the heparin binding site (Church et al, 1989;Karshikov et al, 1992;Arni et al, 1993). The fibrinogen exosite lies between surface loops Leu 33-Leu 41 and Arg 67-Lys 81 with Lys 109-Lys 110 nearby.…”