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J. Biol. Chem., Vol. 282, Issue 40, 29101-29113, October 5, 2007

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Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick^*

Cho Yeow Koh, Maria Kazimirova, Adama Trimnell^¶, Peter Takac, Milan Labuda, Patricia A. Nuttall^||, and R. Manjunatha Kini^**1

From the Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Singapore, the Institute of Zoology, Slovak Academy of Sciences, Dúbravská Cesta 9, Bratislava SK-84506, Slovakia, the ^¶Seattle Biomedical Research Institute, Seattle, Washington 98109-5219, the ^||Natural Environment Research Council Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, United Kingdom, and the ^**Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 13298

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K_i 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8–14, and the exosite-I binding moiety is within residues 15–32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

Received for publication, July 9, 2007 , and in revised form, August 6, 2007.

^* This work was supported by the Academic Research Fund, the National University of Singapore and by the Slovak Research and Development Agency (Grant APVV-51-004505). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Tel.: 65-6874-5235; Fax: 65-6779-2486; E-mail: dbskinim{at}nus.edu.sg.

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