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.coagulation inhibitor | macromolecular recognition | protease | X-ray structure | catalytic triad

The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

“…Therefore, madanins seemingly exert their inhibitory role by outcompeting the enzyme's natural substrates. This is in striking contrast to the other structurally characterized cysteine-less thrombin inhibitors, variegin [16] and anophelin [15]. The latter eludes proteolytic processing by thrombin by employing a unique reverse-binding mode and by specifically disrupting the enzyme's active site [15].…”

“…This is in striking contrast to the other structurally characterized cysteine-less thrombin inhibitors, variegin [16] and anophelin [15]. The latter eludes proteolytic processing by thrombin by employing a unique reverse-binding mode and by specifically disrupting the enzyme's active site [15]. As for variegin, despite binding to and being processed by thrombin in a manner similar to substrates, the C-terminal cleavage product displays significant affinity for the proteinase and anticoagulant activity [16].…”

“…Due to the acidic nature of thrombin's S1 specificity pocket, conferred by the presence of Asp189 at its bottom, arginine and lysine are thrombin's preferred P1 residues. Accordingly, in the current structure of the madanin-1·thrombin complex, the side chain of madanin-1 residue M Arg54 occupies thrombin's S1 site, with a placement reminiscent of other natural substrates and inhibitors [15], [36], [42]. The guanidinium group of M Arg54 establishes several polar contacts with T Asp189 OD1 and the carbonyl oxygen atoms of T Gly219, T Trp215, and T Phe227, which are strengthened by hydrophobic contacts between the M Arg54 side chain and the T Cys191- T Cys220 disulfide bond.…”

“…Another distinctive feature of madanins is the absence of cysteine residues in their amino acid sequence, placing them in the restricted group of cysteine-less thrombin inhibitors, together with thrombostasin (from the horn fly Haematobia irritans [10]; MEROPS family I64), tsetse thrombin inhibitor (from Glossina morsitans morsitans [11]; MEROPS family I76), chimadanin (from H. longicornis [12]; MEROPS family I72), anophelin (from Anopheles mosquitoes [13]; MEROPS family I77) and variegin (from the tropical bont tick Amblyomma variegatum [14]; MEROPS family I74). The molecular mechanism of action of two of these atypical serine proteinase inhibitors, anophelin and variegin, has been unveiled by the crystallographic three-dimensional structures of their complexes with thrombin [15], [16]. Whereas anophelin and variegin bind tightly to thrombin, madanins were shown to bind to thrombin with low affinity [7].…”

More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis

“…Functionally comparable phenylalanine residues are present not only in hirudins (Fig. 4), but also in several other thrombin-inhibiting peptides like anophelin of Anopheles mosquitos (Figueiredo et al 2012) and variegin of Amblyomma variegatum, the tropical bont tick ). In addition to Phe56, the nonpolar residues Ile59, Pro60, and Leu64 are crucial for hydrophobic interactions between the C-terminal tail of hirudin and thrombin (Krstenansky et al 1987;Betz et al 1991;Priestle et al 1993).…”