IRS-2 from the hard tick Ixodes ricinus belongs to the serpin family of protease inhibitors. It is produced in the salivary glands of the tick and its anti-inflammatory activity suggests that it plays a role in parasite-host interaction. Recombinant IRS-2 prepared by heterologous expression in a bacterial system was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the primitive tetragonal space group P4(3) and diffracted to 1.8 Å resolution. Mass-spectrometric and electrophoretic analyses revealed that IRS-2 was cleaved by contaminating proteases during crystallization. This processing of IRS-2 mimicked the specific cleavage of the serpin by its target protease and resulted in a more stable form (the so-called relaxed conformation), which produced well diffracting crystals. Activity profiling with specific substrates and inhibitors demonstrated traces of serine and cysteine proteases in the protein stock solution.

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Flemingovo nám 2 16610 Praha 6 Czech Republic

See more in PubMed

Andreotti, R., Gomes, A., Malavazi-Piza, K. C., Sasaki, S. D., Sampaio, C. A. & Tanaka, A. S. (2002). Int. Immunopharmacol.2, 557–563. PubMed

Baumann, U., Bode, W., Huber, R., Travis, J. & Potempa, J. (1992). J. Mol. Biol.226, 1207–1218. PubMed

DeLano, W. L. (2002). PyMOL. DeLano Scientific, Palo Alto, California, USA.

Dong, A. et al. (2007). Nature Methods, 4, 1019–1021. PubMed PMC

Horn, M., Nussbaumerova, M., Sanda, M., Kovarova, Z., Srba, J., Franta, Z., Sojka, D., Bogyo, M., Caffrey, C. R., Kopacek, P. & Mares, M. (2009). Chem. Biol.16, 1053–1063. PubMed PMC

Huntington, J. A., Read, R. J. & Carrell, R. W. (2000). Nature (London), 407, 923–926. PubMed

Imamura, S., da Silva Vaz Junior, I., Sugino, M., Ohashi, K. & Onuma, M. (2005). Vaccine, 23, 1301–1311. PubMed

Irving, J. A., Pike, R. N., Lesk, A. M. & Whisstock, J. C. (2000). Genome Res.10, 1845–1864. PubMed

Jancarik, J. & Kim, S.-H. (1991). J. Appl. Cryst.24, 409–411.

Law, R. H., Zhang, Q., McGowan, S., Buckle, A. M., Silverman, G. A., Wong, W., Rosado, C. J., Langendorf, C. G., Pike, R. N., Bird, P. I. & Whisstock, J. C. (2006). Genome Biol.7, 216. PubMed PMC

Loebermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984). J. Mol. Biol.177, 531–557. PubMed

Mandel, C. R., Gebauer, D., Zhang, H. & Tong, L. (2006). Acta Cryst. F62, 1041–1045. PubMed PMC

Matthews, B. W. (1968). J. Mol. Biol.33, 491–497. PubMed

Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. (2006). Acta Cryst. D62, 859–866. PubMed

Nagata, K. (1996). Trends Biochem. Sci.21, 22–26. PubMed

Pemberton, P. A., Stein, P. E., Pepys, M. B., Potter, J. M. & Carrell, R. W. (1988). Nature (London), 336, 257–258. PubMed

Prevot, P. P., Beschin, A., Lins, L., Beaufays, J., Grosjean, A., Bruys, L., Adam, B., Brossard, M., Brasseur, R., Zouaoui Boudjeltia, K., Vanhamme, L. & Godfroid, E. (2009). FEBS J.276, 3235–3246. PubMed

Ray, C. A., Black, R. A., Kronheim, S. R., Greenstreet, T. A., Sleath, P. R., Salvesen, G. S. & Pickup, D. J. (1992). Cell, 69, 597–604. PubMed

Sauer, J. R., McSwain, J. L., Bowman, A. S. & Essenberg, R. C. (1995). Annu. Rev. Entomol.40, 245–267. PubMed

Schick, C., Pemberton, P. A., Shi, G. P., Kamachi, Y., Cataltepe, S., Bartuski, A. J., Gornstein, E. R., Bromme, D., Chapman, H. A. & Silverman, G. A. (1998). Biochemistry, 37, 5258–5266. PubMed

Sugino, M., Imamura, S., Mulenga, A., Nakajima, M., Tsuda, A., Ohashi, K. & Onuma, M. (2003). Vaccine, 21, 2844–2851. PubMed

Vagin, A. & Teplyakov, A. (2000). Acta Cryst. D56, 1622–1624. PubMed

Weiss, M. S. (2001). J. Appl. Cryst.34, 130–135.

Zheng, B., Matoba, Y., Kumagai, T., Katagiri, C., Hibino, T. & Sugiyama, M. (2009). Biochem. Biophys. Res. Commun.380, 143–147. PubMed

Zou, Z., Anisowicz, A., Hendrix, M. J., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E. & Sager, R. (1994). Science, 263, 526–529. PubMed

Conformational transition of the Ixodes ricinus salivary serpin Iripin-4

rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick Hyalomma dromedarii

Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus

Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement

Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro

Protease Inhibitors in Tick Saliva: The Role of Serpins and Cystatins in Tick-host-Pathogen Interaction

Ixodes ricinus salivary serpin IRS-2 affects Th17 differentiation via inhibition of the interleukin-6/STAT-3 signaling pathway

A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation