PNAS PLUS

Unique thrombin inhibition mechanism by anophelin,

an anticoagulant from the malaria vector
Ana C. Figueiredoa, Daniele de Sanctisb, Ricardo Gutirrez-Gallegoc,d, Tatiana B. Cereijaa, Sandra Macedo-Ribeiroa,
Pablo Fuentes-Priore, and Pedro Jos Barbosa Pereiraa,1
a
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4150-180 Porto, Portugal; bEuropean Synchrotron Radiation Facility (ESRF), Structural
Biology 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 Biomedical
Research, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain

orientation for blocking enzymatic activity.

Numerous animal species, from insects
(mosquitoes) to mammals (vampire bats),
Anophelin, in contrast, is completely defeed primarily or exclusively on fresh
void of cysteine residues and displays
blood from their prey. These parasites
considerable exibility in solution. The
produce some of the most potent antago3D structure of the anophelinthrombin
nists of the blood clotting system known,
complex revealed an unforeseen reverse
which are critical for their hematophagous
orientation of the inhibitor, which binds to
lifestyle. Many of these compounds are
the proteinase surface in a direction opsmall polypeptides that inhibit the proposite to the direction of natural subteolytic enzymes of the clotting cascade,
strates, although it preserves the majority
notably thrombin. Anopheles mosquitoes
of the molecular contacts established beare widespread hematophagous parasites
tween thrombin and bona de substrates
and important vectors of malaria, a po(Fig. P1). In addition to blocking physical
tentially lethal disease that affects 500
access to thrombins active site, anophelin
million humans worldwide. Here, we show
also impairs enzymatic activity by disruptthat a salivary polypeptide from Anopheles,
ing the characteristic chargerelay system
anophelin, inhibits thrombin by binding
through specic interactions with two of
in an orientation opposite to the orientathe residues required for catalysis. The
tion of substrates and disrupts thrombins
extent of the interactions and the chemical
catalytic machinery (Fig. P1). This
Fig. P1. The malaria vector uses a unique
and structural complementarity between
unique molecular mechanism has implianticoagulant strategy. The crucial procoagulant
thrombin and anophelin explain the antienzyme, thrombin (gray ellipsoid), has two positively
cations for the design of synthetic
coagulant activity of this inhibitor. Anocharged surface regions (blue) important for the
antithrombotics.
phelins unique mechanism of action, now
interaction with substrates and other macromoThe overwhelming majority of prounveiled, has been conserved during the
lecular partners. Natural procoagulant substrates
teinaceous inhibitors of proteolytic
evolution of New and Old World mos(e.g., brinogen) bind across thrombins active site
enzymes works by physically blocking acquitoes, and it can help in the conception
(dashed rectangle) and are cleaved between the P1
cess of the substrate to the active site, and
of more effective and specic thrombin
and P1 residues, leading to thrombus formation
in most cases, they mimic the interactions
(Left). Conversely, anophelin binds to thrombin in
inhibitors with potential application
established by natural substrates. In the
an unexpected reverse orientation (relative to
as antithrombotics.
resulting proteinaseinhibitor complexes,
natural substrates), blocking its catalytic activity in
a unique way and effectively impairing blood
extensive contacts form between the target
1. Bode W, Huber R (2000) Structural basis of the
coagulation (Right). N and C denote the N and C
enzyme and compact and ordered regions
endoproteinase-protein inhibitor interaction. Biochim
termini
of
the
polypeptide
chain,
respectively.
Biophys Acta 1477(12):241252.
of the inhibitors, explaining the high af2. Koh CY, et al. (2011) Crystal structure of thrombin in
nity of the interactions (1). Some known
complex with S-variegin: Insights of a novel mechathrombin inhibitors lack intramolecular covalent links and possess
nism of inhibition and design of tunable thrombin inhibitors. PLoS One 6(10):
unique sequences rich in polar residues, which are likely to be
e26367.
3. Valenzuela JG, Francischetti IM, Ribeiro JM (1999) Purication, cloning, and synthesis of
intrinsically disordered in isolation. Although thrombin cleaves
a novel salivary anti-thrombin from the mosquito Anopheles albimanus. Biochemistry
some of these molecules (2), we found it not to be the case for
38(34):1120911215.
anophelin (MEROPS family I77). Anophelin is an efcient
4. Ronca R, et al. (2012) The Anopheles gambiae cE5, a tight- and fast-binding thrombin
anticoagulant previously identied in two distinct species of
inhibitor with post-transcriptionally regulated salivary-restricted expression. Insect
Anophelesthe Central American A. albimanus (3) and the
Biochem Mol Biol 42(9):610620.
African A. gambiae (4). Here, we performed structural and functional analyses of anophelins. We started by producing anophelins
from all described sources of the anticoagulant and observed
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.,
that, despite their relatively low sequence conservation, all beand P.J.B.P. analyzed data; and A.C.F., S.M.-R., P.F.-P., and P.J.B.P. wrote the paper.
haved as efcient thrombin inhibitors. Biochemical and biophysical
The authors declare no conict of interest.
characterizations of mutated anophelins identied a highly
This article is a PNAS Direct Submission.
conserved region essential for anophelins inhibitory activity. X-ray
Data deposition: The crystallographic atomic coordinates and structure factors have been
crystallographic studies unveiled the molecular mechanism of acdeposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4E05 and 4E06).
tion of the anophelin from A. albimanus. Most structurally char1
To whom correspondence should be addressed. E-mail: ppereira@ibmc.up.pt.
acterized natural serine proteinase inhibitors form highly disuldeSee full research article on page E3649 of www.pnas.org.
linked cores that engage in extensive contacts with the target
Cite this Author Summary as: PNAS 10.1073/pnas.1211614109.
enzyme, positioning the inhibitory segments in the appropriate
www.pnas.org/cgi/doi/10.1073/pnas.1211614109

PNAS | December 26, 2012 | vol. 109 | no. 52 | 21191

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