Selective Inhibition of the Collagenolytic Activity of Human Cathepsin K by Altering Its S2 Subsite Specificity † Fabien Lecaille, ‡ Youngchool Choe, § Wolfgang Brandt, | Zhenqiang Li, ‡ Charles S. Craik, § and Dieter Bro ¨mme* ,‡ Department of Human Genetics, Mount Sinai School of Medicine, Box 1498, New York, New York 10029, Department of Pharmaceutical Chemistry, Program in Chemistry and Chemical Biology and Graduate Group in Biophysics, UniVersity of California San Francisco, San Francisco, California 94143, and Institute of Plant Biochemistry, Martin Luther UniVersity Halle-Wittenberg, Halle/Saale, Germany ReceiVed February 5, 2002; ReVised Manuscript ReceiVed April 29, 2002 ABSTRACT: The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity. Type I collagen is the major component of the organic bone matrix which is continually degraded and resynthesized during the bone remodeling process (1). It consists of covalently cross-linked triple helices containing two R1(I) and one R2(I) chains. Large regions of the collagen chains are composed of repeating Gly-Pro-Xaa sequences, where Xaa is mainly proline and/or 4-trans-L-hydroxyproline (2). Degradation of the triple helical type I collagen is achieved by several members of the matrix metalloprotease family (MMPs) such as MMPs 1, 2, 8, and 13 (3) and the papain- like cysteine protease, cathepsin K (4). MMPs selectively cleave collagens through a single scission across all three chains and generate about three-fourth and one-fourth length collagen fragments (5) whereas cathepsin K, similar to bacterial collagenases, cleaves type I and II collagens at multiple sites within their triple helical domains (6, 7). In contrast, other cysteine proteases such as cathepsins L and B cleave collagens only in their nonhelical telopeptide regions (8). Cathepsin K is predominantly expressed in osteoclasts and has been implicated in bone resorption (9-11). The specific role of cathepsin K in bone resorption was demonstrated by the discovery that deficiency in cathepsin K activity causes an inherited autosomal recessive bone dysplasia, pycno- dysostosis (12). Several cleavage sites of cathepsin K in the triple helical regions of type I and II collagens have been identified (6, 7). The foremost feature of those sites was the frequent presence of proline residues in the P2 position. The accommodation of a P2 proline residue in the S2 subsite pocket [subsite nomenclature from Schechter and Berger (13)] is unique for cathepsin K and in contrast to the preferred acceptance of bulky aromatic P2 residues by cathepsin L. Comparison of three-dimensional structures of cathepsins K and L [PDB, 1mem (14); PDB, 1cjl (15)] showed amino acid sequence differences at residues 67 and 205 in the primary S2 binding pocket (papain numbering is used in this paper). In cathepsin L, residues 67 and 205 are leucine and alanine, respectively, whereas tyrosine and leucine residues are present at the respective positions in cathepsin K. To further explore this unusual preference for proline and to test the hypothesis that residues Y67 and L205 are critical for the ability of cathepsin K to cleave triple helical collagen, a cathepsin L-like double mutant, Tyr67Leu/Leu205Ala, of cathepsin K was generated. The subsite specificities of mutant cathepsin K and wild-type cathepsins K and L using combinatorial peptide libraries were compared, and the ability of the mutant protease to cleave triple helical collagens and gelatin was studied. † This research was supported by NIH Grant AR46182, by a Biomedical Science grant from the Arthritis Foundation, and a postdoctoral scholarship from the Region Centre (France). * To whom correspondence should be addressed. Phone: 212-659- 6753. Fax: 212-849-2508. E-mail: Dieter.Bromme@mssm.edu. ‡ Mount Sinai School of Medicine. § University of California San Francisco. | Martin Luther University. 8447 Biochemistry 2002, 41, 8447-8454 10.1021/bi025638x CCC: $22.00 © 2002 American Chemical Society Published on Web 06/04/2002