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Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases.
Biochemistry. 2003 Sep 30; 42(38):11326-33.B

Abstract

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.

Authors+Show Affiliations

Pavlova A
Department of Molecular Biosciences, Section of Veterinary Medical Biochemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, SE-751 23 Uppsala, Sweden.
Björk I
No affiliation info available

MeSH

Amino Acid SequenceAmino Acid SubstitutionBinding SitesCathepsin BCystatinsCysteine EndopeptidasesCysteine Proteinase InhibitorsHumansKineticsMolecular Sequence DataPapainProtein BindingRecombinant ProteinsSubstrate Specificity

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

14503883

Citation

Pavlova, Alona, and Ingemar Björk. "Grafting of Features of Cystatins C or B Into the N-terminal Region or Second Binding Loop of Cystatin a (stefin A) Substantially Enhances Inhibition of Cysteine Proteinases." Biochemistry, vol. 42, no. 38, 2003, pp. 11326-33.
Pavlova A, Björk I. Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases. Biochemistry. 2003;42(38):11326-33.
Pavlova, A., & Björk, I. (2003). Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases. Biochemistry, 42(38), 11326-33.
Pavlova A, Björk I. Grafting of Features of Cystatins C or B Into the N-terminal Region or Second Binding Loop of Cystatin a (stefin A) Substantially Enhances Inhibition of Cysteine Proteinases. Biochemistry. 2003 Sep 30;42(38):11326-33. PubMed PMID: 14503883.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases. AU - Pavlova,Alona, AU - Björk,Ingemar, PY - 2003/9/25/pubmed PY - 2003/10/30/medline PY - 2003/9/25/entrez SP - 11326 EP - 33 JF - Biochemistry JO - Biochemistry VL - 42 IS - 38 N2 - Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/14503883/Grafting_of_features_of_cystatins_C_or_B_into_the_N_terminal_region_or_second_binding_loop_of_cystatin_A__stefin_A__substantially_enhances_inhibition_of_cysteine_proteinases_ DB - PRIME DP - Unbound Medicine ER -
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