Residue-specific annotation of disorder-to-order transition and cathepsin inhibition of a propeptide-like crammer from D. melanogaster.
Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of 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 removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL) of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
Tien-Sheng
TS
Tseng
Chao-Sheng
CS
Cheng
Shang-Te Danny
ST
Hsu
Min-Fang
MF
Shih
Pei-Lin
PL
He
Ping-Chiang
PC
Lyu
D000409:Alanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002942:Circular Dichroism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D000596:Amino Acids; D015853:Cysteine Proteinase Inhibitors; D029721:Drosophila Proteins; C526367:cer protein, Drosophila; D002401:Cathepsin B; D000409:Alanine