Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain.
Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit mu- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and deltaL110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of Ki = 188 nM. Deletion of L110, which forms a beta-bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu-calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu-calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold.
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik Innenstadt, Klinikum der Ludwig-Maximilians-Universität, München, Germany.
B G
BG
Díaz
S
S
Gross
I
I
Assfalg-Machleidt
D
D
Pfeiler
N
N
Gollmitzer
D
D
Gabrijelcic-Geiger
M T
MT
Stubbs
H
H
Fritz
E A
EA
Auerswald
W
W
Machleidt
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D002645:Chickens / Q000378:metabolism*; D003001:Cloning, Molecular; D055313:Cystatin B; D015891:Cystatins / Q000235:genetics* / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D007704:Kininogens / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
C524994:CSTB protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D007704:Kininogens; D011993:Recombinant Fusion Proteins; D055313:Cystatin B; D002154:Calpain