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
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1108938
- author
- Hall, Anders ; Håkansson, Katarina LU ; Mason, Robert W ; Grubb, Anders LU and Abrahamson, Magnus LU
- organization
- publishing date
- 1995
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 270
- issue
- 10
- pages
- 5115 - 5121
- publisher
- American Society for Biochemistry and Molecular Biology
- 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
- 2016-04-01 11:47:39
- date last changed
- 2021-03-07 05:59:17
@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 = {{American Society for Biochemistry and Molecular Biology}}, 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}}, url = {{http://www.jbc.org/cgi/content/full/270/10/5115}}, volume = {{270}}, year = {{1995}}, }