Abstract
Drosophila melanogaster crammer is a small peptide with 79 amino acids, which involves in long-term memory formation through cathepsin regulation. Although the 3D structure of crammer has been reported, the detailed regulatory mechanism in fruit fly is still unclear. In this study, a site directed mutagenesis approach coupled with biochemical and biophysical methods were used to explore potential roles of the salt bridges in crammer. Alanine substitutions at E8A, Arg28 and Arg29 apparently reduce the thermal stability and alter the protein folding, thus losing their cathepsin inhibitory activities. According to structural analysis, the substitution at Glu8 causes the structural instability of helix-1. Moreover, Arg29 makes close contacts with Asp6 and Asp25 to stabilize helices 1 and 2. These two helices act as an essential scaffold for maintaining the hydrophobic core. Moreover, Arg28 lies on the center of a tri-salt bridge network (Glu24-Arg28-Glu67). This network connects helices 2 and 4 to stabilize the C-terminal orientation of crammer, and to maintain the inhibitory potency of crammer. Accordingly, we have already identified the hot spot residues in crammer, which allows us to expand the potential application for pharmaceutical therapy in the Alzheimer’s disease.