Unique thrombin inhibition mechanism by anophelin,an anticoagulant from the malaria vectorAna C. Figueiredoa, Daniele de Sanctisb, Ricardo Gutirrez-Gallegoc,d, Tatiana B. Cereijaa, Sandra Macedo-Ribeiroa,Pablo Fuentes-Priore, and Pedro Jos Barbosa Pereiraa,1aInstituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4150-180 Porto, Portugal; bEuropean Synchrotron Radiation Facility (ESRF), StructuralBiology Group, 38043 Grenoble Cedex, France; cBioanalysis Group, Neurosciences Research Program, Hospital del Mar Medical Research Institute (IMIM)-Parque de Salud Mar and dDepartment of Experimental and Health Sciences, Pompeu Fabra University, 08003 Barcelona, Spain; and eInstitute for BiomedicalResearch, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain

AUTHOR SUMMARY

Numerous animal species, from insects(mosquitoes) to mammals (vampire bats),feed primarily or exclusively on freshblood from their prey. These parasitesproduce some of the most potent antago-nists of the blood clotting system known,which are critical for their hematophagouslifestyle. Many of these compounds aresmall polypeptides that inhibit the pro-teolytic enzymes of the clotting cascade,notably thrombin. Anopheles mosquitoesare widespread hematophagous parasitesand important vectors of malaria, a po-tentially lethal disease that affects 500million humans worldwide. Here, we showthat a salivary polypeptide from Anopheles,anophelin, inhibits thrombin by bindingin an orientation opposite to the orienta-tion of substrates and disrupts thrombinscatalytic machinery (Fig. P1). Thisunique molecular mechanism has impli-cations for the design of syntheticantithrombotics.The overwhelming majority of pro-

teinaceous inhibitors of proteolyticenzymes works by physically blocking ac-cess of the substrate to the active site, andin most cases, they mimic the interactionsestablished by natural substrates. In theresulting proteinaseinhibitor complexes,extensive contacts form between the targetenzyme and compact and ordered regionsof the inhibitors, explaining the high af-nity of the interactions (1). Some knownthrombin inhibitors lack intramolecular covalent links and possessunique sequences rich in polar residues, which are likely to beintrinsically disordered in isolation. Although thrombin cleavessome of these molecules (2), we found it not to be the case foranophelin (MEROPS family I77). Anophelin is an efcientanticoagulant previously identied in two distinct species ofAnophelesthe Central American A. albimanus (3) and theAfrican A. gambiae (4). Here, we performed structural and func-tional analyses of anophelins. We started by producing anophelinsfrom all described sources of the anticoagulant and observedthat, despite their relatively low sequence conservation, all be-haved as efcient thrombin inhibitors. Biochemical and biophysicalcharacterizations of mutated anophelins identied a highlyconserved region essential for anophelins inhibitory activity. X-raycrystallographic studies unveiled the molecular mechanism of ac-tion of the anophelin from A. albimanus. Most structurally char-acterized natural serine proteinase inhibitors form highly disulde-linked cores that engage in extensive contacts with the targetenzyme, positioning the inhibitory segments in the appropriate

orientation for blocking enzymatic activity.Anophelin, in contrast, is completely de-void of cysteine residues and displaysconsiderable exibility in solution. The3D structure of the anophelinthrombincomplex revealed an unforeseen reverseorientation of the inhibitor, which binds tothe proteinase surface in a direction op-posite to the direction of natural sub-strates, although it preserves the majorityof the molecular contacts established be-tween thrombin and bona de substrates(Fig. P1). In addition to blocking physicalaccess to thrombins active site, anophelinalso impairs enzymatic activity by disrupt-ing the characteristic chargerelay systemthrough specic interactions with two ofthe residues required for catalysis. Theextent of the interactions and the chemicaland structural complementarity betweenthrombin and anophelin explain the anti-coagulant activity of this inhibitor. Ano-phelins unique mechanism of action, nowunveiled, has been conserved during theevolution of New and Old World mos-quitoes, and it can help in the conceptionof more effective and specic thrombininhibitors with potential applicationas antithrombotics.

1. Bode W, Huber R (2000) Structural basis of theendoproteinase-protein inhibitor interaction. BiochimBiophys Acta 1477(12):241252.

2. Koh CY, et al. (2011) Crystal structure of thrombin incomplex with S-variegin: Insights of a novel mecha-

nism of inhibition and design of tunable thrombin inhibitors. PLoS One 6(10):e26367.

3. Valenzuela JG, Francischetti IM, Ribeiro JM (1999) Purication, cloning, and synthesis ofa novel salivary anti-thrombin from the mosquito Anopheles albimanus. Biochemistry38(34):1120911215.

4. Ronca R, et al. (2012) The Anopheles gambiae cE5, a tight- and fast-binding thrombininhibitor with post-transcriptionally regulated salivary-restricted expression. InsectBiochem Mol Biol 42(9):610620.

Author contributions: A.C.F., D.d.S., R.G.-G., S.M.-R., P.F.-P., and P.J.B.P. designed research;A.C.F., D.d.S., R.G.-G., and T.B.C. performed research; A.C.F., D.d.S., R.G.-G., S.M.-R., P.F.-P.,and P.J.B.P. analyzed data; and A.C.F., S.M.-R., P.F.-P., and P.J.B.P. wrote the paper.

The authors declare no conict of interest.

This article is a PNAS Direct Submission.

Data deposition: The crystallographic atomic coordinates and structure factors have beendeposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4E05 and 4E06).1To whom correspondence should be addressed. E-mail: ppereira@ibmc.up.pt.

See full research article on page E3649 of www.pnas.org.

Cite this Author Summary as: PNAS 10.1073/pnas.1211614109.

Fig. P1. The malaria vector uses a uniqueanticoagulant strategy. The crucial procoagulantenzyme, thrombin (gray ellipsoid), has two positivelycharged surface regions (blue) important for theinteraction with substrates and other macromo-lecular partners. Natural procoagulant substrates(e.g., brinogen) bind across thrombins active site(dashed rectangle) and are cleaved between the P1and P1 residues, leading to thrombus formation(Left). Conversely, anophelin binds to thrombin inan unexpected reverse orientation (relative tonatural substrates), blocking its catalytic activity ina unique way and effectively impairing bloodcoagulation (Right). N and C denote the N and Ctermini of the polypeptide chain, respectively.

www.pnas.org/cgi/doi/10.1073/pnas.1211614109 PNAS | December 26, 2012 | vol. 109 | no. 52 | 21191

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