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

Abstract

Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized d-Phe-Pro-Arg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E(ht), corresponding to the central region of human fibrin, and solved its structure at 3.65-A resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E(ht) in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E(ht). Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A alpha chain Trp-33, Phe-35, Asp-38, Glu-39, the B beta chain Ala-68 and Asp-69, and the gamma chain Asp-27 and Ser-30 of E(ht) form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.

Fig. 1.

Two different projections of the three-dimensional structure of the complex of thrombin with the E_ht fragment. (A and B) Ribbon diagram and solvent-accessible surface of the complex viewed along a noncrystallographic twofold symmetry axis perpendicular to the plane of the page, respectively. (C) The complex viewed along the axial projection of the coiled-coil domains. Aα, Bβ, and γ chains in A are in blue, green, and red, respectively. NH₂-terminal portions of the Aα and Bβ chains and that of the γ chain forming the funnel-shaped and the γN-domains, respectively (33), in B and C have the same color scheme as in A, whereas their remaining portions forming the coiled coil domains are in gray. Thrombin molecules in all panels are in beige, whereas the PPACK inhibitor bound to the active sites is in magenta and residues included in exosite I (16) are in orange. The distance between the active sites is ≈70 Å. The broken curved lines in B indicate the S' groove in the active site cleft of each thrombin molecule connecting the active site with exosite I.

Fig. 2.

Stereoview of the hydrophobic interior of the interface between thrombin and the E_ht fragment and the surrounding network of polar interactions. Residues forming the interior are covered with wire mesh van der Waals atomic surfaces and colored as their parent chains, beige for thrombin and blue, green, and red for the Aα,Bβ and γ chains of E_ht, respectively. Residues involved in the polar interactions are colored by atom types: blue for nitrogens, red for oxygens, and white for carbons; interatomic contacts are represented with dashed lines. The colors of the labels coincide with those of the corresponding chains. A view of the whole interface region is published as supporting information on the PNAS web site.

Fig. 3.

Location of the complementary binding sites in the E_ht fragment and thrombin. (Upper) Residues of E_ht involved in the interaction with thrombin, Aα chain Trp-33, Phe-35, Asp-38, Glu-39, Bβ chain Ala-68 and Asp-69 of the thrombin-binding domain, and γ chain Asp-27 and Ser-30 of the coiled-coil domain (in gray). AαSer-37, which could also participate in the interaction (see text), and AαAsp-40 and BβAsp-71, of the proposed negatively charged platform (34), are presented as well. The residues are presented in a projection similar to that for the corresponding residues in Fig. 2. (Lower) Thrombin residues interacting with E_ht, Phe-34, Ser-36A, Leu-65, Ile-82, Tyr-76, Arg-77A, and Lys-110; residues included in exosite I (16) are also highlighted. In both panels, residues are colored according to their properties: hydrophobic residues are in yellow, positively and negatively charged residues are in blue and red, respectively, and polar residues as well as AαTrp-33 forming polar contact are in pink. The complementary binding surfaces in both molecules are contoured with triangles; the repeating end arrows connecting the vertices show their mutual orientations in the complex.