Figure 1.
Structure of thrombin:s-variegin complex.
(A) Thrombin (yellow) shown in the classical orientation in ribbon (without s-variegin). Side chains of catalytic triad, TAsp102, THis57 and TSer195 are shown in sticks (green). The 60-loop, autolysis loop and Na+-binding loop are circled in brown, cyan and green, respectively. Parts of thrombin forming the anion-binding exosite-I and exosite-II are circled in blue and purple, respectively. (B) Surface representation of thrombin (yellow) in the same orientation as (a). Locations of active site specificity pocket, non-prime and prime subsites are indicated by arrows. (C) The structure of thrombin (yellow) in the same orientation as above shown in complex with s-variegin (pink) together with its electron density map (2Fo-Fc) shown contoured at 0.9σ. (D) Surface representation of thrombin in complex with s-variegin (pink).
Table 1.
Crystallographic data and refinement statistics.
Figure 2.
Thrombin catalytic triads in s-variegin-bound and hirugen-bound structures.
(A) Thrombin catalytic triad THis57, TAsp102 and TSer195 in thrombin:hirugen structure (green) and in thrombin:s-variegin structure (pink) are superimposed. The TSer195 Oγ in thrombin:s-variegin structure is displaced by 1.19 Å compared to thrombin:hirugen structure. The displacement of TSer195 Oγ in thrombin:s-variegin structure (pink) is due to interactions with VHis12 of s-variegin through hydrogen bond (dotted arrow), rendering TSer195 a weak nucleophile that is incapable of catalysis. The imidazole ring of THis57 also rotated, resulted in a displacement of its Nε by 0.56 Å. Overall, the distance between Nε of THis57 and Oγ of TSer195 increases to 3.60 Å (black arrow) from 2.79 Å (green arrow), disrupts the catalytic charge relay system. (B) The 2Fo-Fc electron density map of thrombin catalytic triad and VHis12 contoured at 1.0σ.
Figure 3.
Interactions between thrombin and s-variegin.
(A) Prime subsites interactions between thrombin and s-variegin (residues P2′ to P5′) are shown. Density for s-variegin P1′ VMet11 cannot be traced in the structure. Thrombin S2′ subsite is colored in red, S3′ subsite in cyan, S4′ subsite in pink and S5′ subsite in green. (B) Thrombin residues that interfaced with s-variegin are colored according to their positions: catalytic pocket (blue): THis57, TCys58, TCys191, TGlu192, TGly193, TSer195; 60-loop (red): TTrp60D and TLys60F; autolysis loop (cyan): TTrp141, TGly142, TAsn143, TThr147 and TGln151; 34-loop (brown): TPhe34, TArg35, TGln38 and TGlu39; 70-loop (green): TArg73, TThr74, TArg75, TTyr76 and TArg77A; bottom of the cleft (orange): TMet32, TLeu40, TLeu41, TCys42, TLeu65, TArg67, TLys81, TIle82, TMet84 and TLys110. Sticks model of s-variegin is shown in pink. (C) All but four residues (VPhe18, VAla22, VGlu25 and VLeu28, white) on s-variegin have their side chains buried in the interface with thrombin.
Figure 4.
Electrostatic interactions in thrombin:s-variegin structure.
(A) s-Variegin and hirulog-3 have distinct ion pairs formed with exosite-I of thrombin despite high sequence identity. A salt bridge (3.84 Å) between VGlu26 (pink) and TArg77A (yellow) is absent in hirulog-3 as TArg77A (cyan) points away from the inhibitor. Weak salt bridge (4.64 Å) is also likely between VGlu21 (pink) and TArg75 (yellow) rotated 90.5° about Cβ compared to TArg75 in hirulog-3 bound thrombin (cyan) to facilitate interaction with VGlu21 (pink). Electron density maps of residues involved are shown in Figure S2A. (B) The strong ion pair (Asp11∶TArg73, 2.92 Å) in thrombin:hirulog-3 structure is absent in thrombin:s-variegin structure since VAsp19 (pink) pointed to an opposite direction compared to the analogous hirulog-3 Asp11 (blue) due to a kink in s-variegin backbone (pink). Electron density maps of residues involved are shown in Figure S2B. (C) The presence of a VPro16-VPro17 (green) in s-variegin resulted in the kink. Superimposition of s-variegin (pink, only Cα positions traced) and hirulog-3 (blue, only Cα positions traced) based on their thrombin structures showed displacement of VPhe18 and VAsp19 from their corresponding residues Gly10 and Asp11 of hirulog-3 by 3.11 Å and 0.79 Å (measured by Cα positions), respectively. As a result, the distance between TArg73 and VAsp19 charges are 5.83 Å, rendering electrostatic interactions impossible.
Table 2.
Sequence and activity of variegin and its variants.
Figure 5.
Ki values of all peptides (including hirulog-1/bivalirudin).
Peptides are grouped according to their mechanism of actions. All competitive inhibitors (fast or slow) have higher affinities to thrombin compared to hirulog-1/bivalirudin. The most potent variant DV24K10RYsulf is about 70-fold stronger. Even their cleavage products (non-competitive inhibitors) are potent inhibitor, with one of them, MH18Ysulf, binds to thrombin approximately 2-fold tighter than hirulog-1/bivalirudin.
Figure 6.
In vivo antithrombotic effects of peptides.
Zebrafish 4 days post-fertilization larvae were injected with 10 nl of different peptides at 500 µM or 10 nl of PBS as control. TTO for larvae injected with PBS, hirulog-1/bivalirudin, s-variegin, EP25 and MH22 are 19.0±3.2 s, 45.0±5.5 s, 120.8±7.4 s, 22.5±6.2 s and 33.3±2.9 s, respectively. Within 150 s, no thrombus was formed in larvae injected with DV24K10RYsulf. With the exception of the slow binding inhibitor EP25, the abilities of the peptides to prolong TTO correlate with their in vitro Ki (n = 4, error bars represent S.D.).