Abstract
Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in long-term memory (LTM) formation. The mechanism by which the inhibitory activity is regulated remains unclear. Here we have shown that at neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, upon binding to cathepsin L; however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that the C72S variant renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of a propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin. In addition, alanine substitution for the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence and nuclear magnetic resonance (NMR) spectroscopies show that the removal of most the aromatic and charged side-chains substantially reduce the thermostability, alter pH-dependent helix formation, and disrupt the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 is essential for the interaction between crammer and CTSB; the salt bridge R28-E67 is critical for the appropriate alignment of the -helix 4 towards the CTSB active cleft. Alanine scanning provides detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges of crammer, and these insights could serve as a template for further development of therapeutic inhibitors against cathepsins.