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The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases

Bjork, Ingemar; Brieditis, Ingrid; Raub-Segall, Elke; Pol, Ewa; Håkansson, Katarina and Abrahamson, Magnus LU (1996) In Biochemistry 35(33). p.10720-10726
Abstract
The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ·mol-1, to the binding to all four cysteine proteinases, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp,... (More)
The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ·mol-1, to the binding to all four cysteine proteinases, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp, showing that a phenyl group can only partly substitute for the indole ring. The reduced affinities of the cystatin C Trp-106 variants for all proteinases studied were due almost exclusively to increased dissociation rate constants. The second hairpin loop thus contributes to the binding primarily by keeping cystatin C anchored to the proteinase once the complex has been formed. This role is partly in contrast to that of the N-terminal region, which increases the affinity of cystatin C for cathepsin B by increasing the association rate constant. Removal of the N-terminal region of the Trp-106Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B. The resulting affinity indicated that the first hairpin loop (the "QVVAG-region"), which is the only region of the proteinase binding surface remaining intact in the truncated variant, contributes 40-60% of the total free energy of binding of cystatin C to both proteinases. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
35
issue
33
pages
10720 - 10726
publisher
The American Chemical Society
external identifiers
  • Scopus:0029776802
ISSN
0006-2960
DOI
10.1021/bi960420u
language
English
LU publication?
yes
id
a6cf3e50-1d38-4393-9c17-94b050ef5fa5 (old id 1111062)
date added to LUP
2008-07-22 08:59:22
date last changed
2017-02-26 04:29:22
@article{a6cf3e50-1d38-4393-9c17-94b050ef5fa5,
  abstract     = {The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ·mol-1, to the binding to all four cysteine proteinases, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp, showing that a phenyl group can only partly substitute for the indole ring. The reduced affinities of the cystatin C Trp-106 variants for all proteinases studied were due almost exclusively to increased dissociation rate constants. The second hairpin loop thus contributes to the binding primarily by keeping cystatin C anchored to the proteinase once the complex has been formed. This role is partly in contrast to that of the N-terminal region, which increases the affinity of cystatin C for cathepsin B by increasing the association rate constant. Removal of the N-terminal region of the Trp-106Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B. The resulting affinity indicated that the first hairpin loop (the "QVVAG-region"), which is the only region of the proteinase binding surface remaining intact in the truncated variant, contributes 40-60% of the total free energy of binding of cystatin C to both proteinases.},
  author       = {Bjork, Ingemar and Brieditis, Ingrid and Raub-Segall, Elke and Pol, Ewa and Håkansson, Katarina and Abrahamson, Magnus},
  issn         = {0006-2960},
  language     = {eng},
  number       = {33},
  pages        = {10720--10726},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases},
  url          = {http://dx.doi.org/10.1021/bi960420u},
  volume       = {35},
  year         = {1996},
}