Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition.
Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by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.
Hall
A
A
Department of Clinical Chemistry, University Hospital, Lund, Sweden.
Dalbøge
H
H
Grubb
A
A
Abrahamson
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003062:Codon; D017124:Conserved Sequence; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
C525030:CST3 protein, human; D003062:Codon; D055316:Cystatin C; D015891:Cystatins; D011994:Recombinant Proteins; D002401:Cathepsin B; D010206:Papain; D005998:Glycine