Inhibition of human mu-calpain by conformationally constrained calpastatin peptides.
The 27-mer peptide CP1B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.
Pfizer
José
J
Max-Planck-Institut für Biochemie, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Assfalg-Machleidt
Irmgard
I
Machleidt
Werner
W
Schaschke
Norbert
N
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors*; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D015195:Drug Design; D006801:Humans; D007700:Kinetics; D007769:Lactams / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D007769:Lactams; D010455:Peptides; C033668:calpastatin; D002154:Calpain; C449002:mu-calpain