Interaction between cystatin-derived peptides and papain.
The interaction between papain and synthetic peptides which tentatively mimic cystatin surfaces was investigated both enzymatically and structurally. Measurements of dissociation equilibrium constants for the interaction of papain with these peptides modified by successive deletions or substitutions demonstrated that the QVVAG segment, which is highly conserved throughout members of the cystatin superfamily, is essential for the interaction. The glycyl-containing (N-terminal) fragments and PW-containing (C-terminal) fragments were found to be of lesser importance, since each could be deleted without significantly modifying the interaction. These fragments improved the stability of the interacting QVVAG region, which appeared to be substrate-like in all peptides tested, as it was cleaved at the A-G bond upon peptide-papain interaction. Replacement of the A residue at the scissile bond of the QVVAG by a blocked cysteinyl residue reduced the rate of cleavage of the susceptible bond and therefore shifted the resulting peptide from a substrate to an inhibitor. Derivatization of this substituted peptide at its N- and C-terminal ends by fluoresceinyl groups resulted in a dramatic decrease in the Ki to 0.5 microM. This improvement in the inhibitory properties of the substituted and derivatized peptides was correlated with structural changes as analyzed by molecular dynamic calculations. The results were compared to those proposed for the mechanisms of inhibition by natural inhibitors of the cystatin superfamily.
Lalmanach
G
G
URA CNRS 1334, University François Rabelais, Faculty of Medicine, Tours, France.
Hoebeke
J
J
Moreau
T
T
Brillard-Bourdet
M
M
Ferrer-Ditt Martino
M
M
Borras-Cuesta
F
F
Gauthier
F
F
D000595:Amino Acid Sequence; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
D015891:Cystatins; D010446:Peptide Fragments; D010206:Papain