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Structural basis for different inhibitory specificities of human cystatins C and D

Hall, Anders; Ekiel, I; Mason, RW; Kasprzykowski, F; Grubb, Anders LU and Abrahamson, Magnus LU (1998) In Biochemistry 37(12). p.4071-4079
Abstract
Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of... (More)
Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D. (Less)
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organization
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Contribution to journal
publication status
published
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in
Biochemistry
volume
37
issue
12
pages
4071 - 4079
publisher
The American Chemical Society
ISSN
0006-2960
language
English
LU publication?
yes
id
ae31d093-c29f-4c9a-b6dd-0b9a10200285 (old id 1113865)
alternative location
http://pubs.acs.org/cgi-bin/article.cgi/bichaw/1998/37/i12/html/bi971197j.html
date added to LUP
2008-07-16 12:52:57
date last changed
2016-04-15 19:34:16
@article{ae31d093-c29f-4c9a-b6dd-0b9a10200285,
  abstract     = {Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.},
  author       = {Hall, Anders and Ekiel, I and Mason, RW and Kasprzykowski, F and Grubb, Anders and Abrahamson, Magnus},
  issn         = {0006-2960},
  language     = {eng},
  number       = {12},
  pages        = {4071--4079},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Structural basis for different inhibitory specificities of human cystatins C and D},
  volume       = {37},
  year         = {1998},
}