Abstract
Drosophila melanogaster crammer is a small protein with 79 amino acids. The primary sequence of crammer is similar to that of the propeptide of cathepsin, therefore, crammer has been considered as a novel propeptide-like inhibitor. Recently, crammer has been reported to involve in the formation of long term memory by regulating the activity of cathepsin, but its regulatory mechanism is still unclear. In this study, site directed mutagenesis was applied to explore the hot spot residues of crammer in inhibitory potency against cathepsin. Meanwhile, biochemical and biophysical methods were used to clarify structural and functional relationships. Our inhibitory assay reveals that the conserved aromatic residues with other organisms in crammer play an important role in inhibitory potency. Alanine substitutions at these positions lose more than 20% inhibitory ability. Moreover, most of mutants have no obvious influences in protein structure, except for W9A, Y12A and Y20A. Trp9 in crammer made close contacts with Y12, F16 and Y20 by pi-pi stacking and alanine replacement disrupts this interaction, thus resulting in the structural unfolded. On the other hand, the functional loss of W53 is resulted from disrupting the interaction between cathepin B and crammer. Taken together, this study identified the hot spot residues of crammer in cathepsin inhibition, which will expand the potential of pharmaceutical theapry Alzheimer’s disease.