Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector

Abstract

Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin. Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32-61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin's active site charge-relay system, and the insertion of residue R53 into the proteinase S(1) pocket in an orientation opposed to productive substrates explain anophelin's remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.

Fig. 1.

Sequence alignment of family I77 inhibitors from anopheline mosquitoes. The mature sequences of anophelins from the Old World mosquito species A. gambiae [pink eye standard (PEST) variant] (62, 63), A. stephensi (1), and A. funestus (3) and the New World species A. albimanus (6, 7) and A. darlingi (2) (vectors of malaria in sub-Saharan Africa, India, Africa, and Central and South America, respectively) were aligned with ClustalW (64). Also included is the sequence of TTI (G. morsitans morsitans; MEROPS family I76) (47). Strictly conserved residues are highlighted in green, whereas other conservative replacements are shaded red. The numbering for mature anophelin^Aa is given above the alignment, and the numbering for mature TTI is given above the respective sequence. The figure was prepared with Aline (65).

Fig. 2.

Anophelin binds with high affinity to and stabilizes human α-thrombin. (A) Thrombin stability to thermal denaturation was measured by DSF in the presence of a 50-fold molar excess of WT anophelins, anophelin^1–31, anophelin^32–61, or anophelin mutants. Melting temperatures (T_m) were determined as the inflection points of the experimental curves, and they are given as mean values ± SEM. (B) Anophelin binding to immobilized human α-thrombin was measured by SPR. Sensorgrams depict kinetics experiments for WT anophelin and anophelin^32–61. Each set of experimental curves (green) represents decreasing concentrations of analyte in twofold dilution steps (the highest concentration used is indicated). The black traces represent the fitted data according to the 1:1 Langmuir binding model. RU, resonance unit.

Fig. 3.

Anophelin inhibits α-thrombin in a unique reverse-binding mode. (A) The acidic ^AE32-^AF45 segment of anophelin (stick model with nitrogen atoms in blue, oxygen in red, and carbon in green) binds to the exosite I of thrombin (positive surface electrostatic potential in blue and negative surface electrostatic potential in red), whereas the downstream ^AD50-^AL55 segment occupies the active site cleft of the proteinase. The unbiased Fo-Fc electron density (1.5-σ cutoff) for anophelin is displayed as a blue mesh. The thrombin molecule is shown in the standard orientation for serine proteinases (i.e., substrates would run from left to right or exactly opposite to the path followed by the Anopheles inhibitor). (B) Close-up view of the Van der Waals interactions between ^AE38 (anophelin colored as in A) and the pivotal ^TY76 at the exosite I of thrombin [gray cartoon with selected residues as sticks color-coded as anophelin except for carbon atoms (colored salmon); active site residues in ball and stick]. (C) Close-up view of the interaction between the strictly conserved ^AD50 and the active site residues ^TH57 and ^TS195 of thrombin (colors as in B). The invariant ^AR53 occupies the S₁ specificity pocket. Water molecules are represented as red spheres. B, Right and C, Right provide schematic representations of the main anophelin⋅thrombin interactions. Hydrogen bonds are represented as dotted black lines. The figure was prepared with PyMOL (http://www.pymol.org) and PoseView (66).

Fig. 4.

Schematic representation of the unique mechanism of thrombin recognition by family I77 inhibitors compared with substrates and other inhibitors. The thrombin molecule is represented as an orange ellipse, with exosites in blue and the active site in red. (A) The natural substrate PAR1 binds to the active site, and the region C terminus to the scissile bond interacts with the exosite I of the proteinase. (B) Thrombin-activated coagulation factors V and VIII bind in a canonical way to the active site of the enzyme, interacting with both exosites through the region upstream to the cleavable bond. (C) The natural anticoagulant from Hirudo medicinalis, hirudin, interacts with the exosite I of thrombin through its C-terminal acidic segment and blocks the active site of the enzyme with its N terminus. (D) MEROPS family I77 inhibitors from the Anopheles mosquitoes bind to thrombin in a reverse orientation relative to natural substrates: the N-terminal portion of anophelin recognizes the exosite I of the enzyme, whereas the C-terminal acidic segment binds to the active site region of the proteinase.

Fig. P1.

The malaria vector uses a unique anticoagulant strategy. The crucial procoagulant enzyme, thrombin (gray ellipsoid), has two positively charged surface regions (blue) important for the interaction with substrates and other macromolecular partners. Natural procoagulant substrates (e.g., fibrinogen) bind across thrombin’s active site (dashed rectangle) and are cleaved between the P1 and P1′ residues, leading to thrombus formation (Left). Conversely, anophelin binds to thrombin in an unexpected reverse orientation (relative to natural substrates), blocking its catalytic activity in a unique way and effectively impairing blood coagulation (Right). N and C denote the N and C termini of the polypeptide chain, respectively.