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

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-a...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2012-12, Vol.109 (52), p.E3649-E3658
Main Authors: Figueiredo, Ana C, de Sanctis, Daniele, Gutiérrez-Gallego, Ricardo, Cereija, Tatiana B, Macedo-Ribeiro, Sandra, Fuentes-Prior, Pablo, Pereira, Pedro José Barbosa
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Anopheles - chemistry
Anticoagulants
Anticoagulants - chemistry
Anticoagulants - pharmacology
Antithrombins - chemistry
Antithrombins - pharmacology
Arginine - metabolism
Binding sites
Biochemistry
Biological Sciences
Blood Coagulation - drug effects
Catalytic Domain
Conserved Sequence
Crystallography, X-Ray
Humans
Immobilized Proteins - metabolism
Insect Proteins - chemistry
Insect Proteins - pharmacology
Insect Vectors - chemistry
Kinetics
Malaria
Malaria - parasitology
Models, Molecular
Molecular Sequence Data
Mosquitoes
Peptides
PNAS Plus
Protein Binding - drug effects
Protein Stability - drug effects
Proteins
Salivary Proteins and Peptides - chemistry
Salivary Proteins and Peptides - pharmacology
Sequence Alignment
Structure-Activity Relationship
Substrate Specificity - drug effects
Surface Plasmon Resonance
Thrombin - antagonists & inhibitors
Thrombin - metabolism
Thrombin Time
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1211614109