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

Hall, Anders; Dalboge, Henrik; Grubb, Anders LU and Abrahamson, Magnus LU (1993) In Biochemical Journal 291(1). p.123-129
Abstract
Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by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... (More)
Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by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. (Less)
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publication status
published
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in
Biochemical Journal
volume
291
issue
1
pages
123 - 129
publisher
Portland Press Limited
ISSN
0264-6021
language
English
LU publication?
yes
id
1a15107a-6d98-4fe6-913c-516e093482be (old id 1107625)
alternative location
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1132490
date added to LUP
2008-07-31 13:45:35
date last changed
2016-04-16 03:57:04
@article{1a15107a-6d98-4fe6-913c-516e093482be,
  abstract     = {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.},
  author       = {Hall, Anders and Dalboge, Henrik and Grubb, Anders and Abrahamson, Magnus},
  issn         = {0264-6021},
  language     = {eng},
  number       = {1},
  pages        = {123--129},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Importance of the evolutionarily conserved glycine residue in the N-terminal region of cystatin C (Gly-11) for cysteine endopeptidase inhibition},
  volume       = {291},
  year         = {1993},
}