Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition

• Published in: Biochemical journal 1993-04, Vol.291 (1), p.123-129
• Main Authors: HALL, A, DALBØGE, H, GRUBB, A, ABRAHAMSON, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Cathepsin B - antagonists & inhibitors; Cathepsin B - metabolism; Chemical Phenomena; Chemistry, Physical; Codon; Conserved Sequence; Cystatin C; Cystatins - chemistry; Cystatins - genetics; Cystatins - pharmacology; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Glycine - chemistry; Humans; Hydrolases; Molecular Sequence Data; Mutagenesis, Site-Directed; Papain - antagonists & inhibitors; Papain - metabolism; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2910123

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.