Inspection of ion switch residues that mediate the pH-dependent conformational change of 'short' recombinant human pseudocathepsin D

Cathepsin D (CatD) is a major intracellular aspartyl peptidase normally found in the endosomes and lysosomes of higher eukaryotes. Traditionally, CatD was believed to function mainly in protein turnover in the lysosome. However, it also functions in vital processes including prohormone and antigen processing and in the generation of biologically active molecules such as angiostatin.{09}In addition to its important physiological roles, CatD has been implicated in a number of pathological events including breast cancer and gastric carcinomas. In light of its low pH optimum, it is interesting that CatD has been isolated from extra-lysosomel locations including the cytosol and extracellular matrix where the pH is more neutral.; High resolution X-ray crystal structures have been solved for an active low pH (pH 5.1) form (CatD_lo) and an inactive high pH (pH 7.5) form (CatD_hi). Comparison of CatD_hi and CatD_lo reveals significant conformational differences. Moreover, ionizable switches may mediate the reversible interconversion between the active and inactive forms. The work presented here focused on delineating the function of specific residues believed to play a role in the pH-dependent conformational change of CatD.; We used site-directed mutagenesis to change the amino acids suggested to mediate the pH-dependent conformational change to neutral residues. Kinetic and structural characterization of the variants indicated that glutamic acid 180 and aspartic acid 187 interact as an ionizable switch. Charge repulsion from the ionization of the opposing carboxylates due to an increase in pH initiates the pH-dependent conformational change of active CatD_lo to inactive CatD_hi. Additionally, tyrosine 10 stabilizes CatD _hi by hydrogen bonding to the catalytic aspartic acid 32. Finally, glutamic acid 5 stabilizes CatD_lo through its hydrogen bonding interaction with glutamic acid 18. This appears to play a less significant role in mediating the conformational change.; This information will contribute to the understanding of various cellular location-dependent activities associated with CatD. Additionally, this study has resulted in the discovery of CatD variants with modified conformational equilibria that may be useful in gene therapy strategies. This information may be useful in the design of novel inhibitors.