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.
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.
authors.affiliation: Department of Clinical Chemistry, University of Lund, University Hospital, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Hall
authors.forename: K
authors.initials: K
authors.lastname: Håkansson
authors.forename: R W
authors.initials: RW
authors.lastname: Mason
authors.forename: A
authors.initials: A
authors.lastname: Grubb
authors.forename: M
authors.initials: M
authors.lastname: Abrahamson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D055316:Cystatin C; D015891:Cystatins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D017931:DNA Primers; D004355:Drug Stability; D004926:Escherichia coli; D005998:Glycine; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D015183:Restriction Mapping; D013816:Thermodynamics
chemical_list: C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D005998:Glycine