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PMID:7890620

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Citation

Hall, A, Håkansson, K, Mason, RW, Grubb, A and Abrahamson, M (1995) 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. J. Biol. Chem. 270:5115-21

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.

Links

PubMed

Keywords

Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Biological Evolution; Cathepsins/antagonists & inhibitors; Cloning, Molecular; Conserved Sequence; Cystatin C; Cystatins/biosynthesis; Cystatins/chemistry; Cystatins/pharmacology; Cysteine Endopeptidases/metabolism; Cysteine Proteinase Inhibitors/pharmacology; DNA Primers; Drug Stability; Escherichia coli; Glycine; Humans; Mammals; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/pharmacology; Restriction Mapping; Thermodynamics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:CYTC

involved_in

GO:0010716: negative regulation of extracellular matrix disassembly

ECO:0000305: curator inference used in manual assertion

GO:0010466

P

Seeded From UniProt

complete

HUMAN:CYTC

involved_in

GO:0010466: negative regulation of peptidase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:CYTC

enables

GO:0004869: cysteine-type endopeptidase inhibitor activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:CYTC

enables

GO:0002020: protease binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P07858

F

Seeded From UniProt

complete

HUMAN:CATB

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P01034

F

Seeded From UniProt

complete

HUMAN:CATB

enables

GO:0008233: peptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:CATB

involved_in

GO:0006508: proteolysis

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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