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Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases

Hall, Anders; Håkansson, Katarina LU ; Mason, Robert W; Grubb, Anders LU and Abrahamson, Magnus LU (1995) In Journal of Biological Chemistry 270(10). p.5115-5121
Abstract
The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated,... (More)
The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H. (Less)
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author
organization
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Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
270
issue
10
pages
5115 - 5121
publisher
ASBMB
external identifiers
  • pmid:7890620
  • scopus:0028968170
ISSN
1083-351X
language
English
LU publication?
yes
id
53c32629-be88-4082-9879-46d4b5686cc1 (old id 1108938)
alternative location
http://www.jbc.org/cgi/content/full/270/10/5115
date added to LUP
2008-07-25 11:21:49
date last changed
2017-08-06 03:34:33
@article{53c32629-be88-4082-9879-46d4b5686cc1,
  abstract     = {The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.},
  author       = {Hall, Anders and Håkansson, Katarina and Mason, Robert W and Grubb, Anders and Abrahamson, Magnus},
  issn         = {1083-351X},
  language     = {eng},
  number       = {10},
  pages        = {5115--5121},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases},
  volume       = {270},
  year         = {1995},
}