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J. Biol. Chem., Vol. 283, Issue 15, 9896-9908, April 11, 2008

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Structural and Functional Relationships in the Virulence-associated Cathepsin L Proteases of the Parasitic Liver Fluke, Fasciola hepatica^*

Colin M. Stack¹, Conor R. Caffrey, Sheila M. Donnelly, Amritha Seshaadri, Jonathan Lowther, Jose F. Tort^¶, Peter R. Collins², Mark W. Robinson³, Weibo Xu, James H. McKerrow, Charles S. Craik^||, Sebastian R. Geiger^**4, Rachel Marion^**, Linda S. Brinen^**, and John P. Dalton, Recipient of the New South Wales Government BioFirst Award in Biotechnology⁵

From the Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Sydney, New South Wales 2007, Australia, Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, California 94158, the ^¶Departamento de Genetica, Facultad de Medicina, Universidad del la Republica, General Flores 2125, CP 11800, Montevideo, Uruguay, the ^||Departments of Pharmaceutical Chemistry, Pharmacology, and Biochemistry and Biophysics, University of California, San Francisco, California 94158, and the ^**Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-Å three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

Received for publication, October 15, 2007 , and in revised form, November 28, 2007.

The atomic coordinates and structure factors (code 2O6X) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by The Sandler Family Supporting Foundation. 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.

¹ Supported by the Australian Research Council. Present address: Dept. of Medical Microbiology, University of Western Sydney, Narellan Rd., Campbelltown, New South Wales, Australia.

² Supported by a grant received from Enterprise Ireland and Dept. of Science, Education, and Training, Australia. Present address: The School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland.

³ Recipient of a Wain International Travel Scholarship, UK.

⁴ Present address: Gene Center Munich, Dept. of Chemistry and Biochemistry, Ludwig-Maximilians-Universitat Munchen, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany.

⁵ To whom correspondence should be addressed: Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Level 6, Bldg. 4, Corner of Thomas & Harris St., Ultimo, Sydney, New South Wales 2007, Australia. Tel.: 61-2-9514-4142; Fax: 61-2-9514-4201; E-mail: john.dalton{at}uts.edu.au.

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