title: The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73.
abstract: The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by approximately 300-fold, >10-fold and approximately 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by approximately 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, approximately 45% of the total binding energy, for inhibition of cathepsin B.
authors.affiliation: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden.
authors.forename: Alona
authors.initials: A
authors.lastname: Pavlova
authors.forename: Ingemar
authors.initials: I
authors.lastname: Björk
mesh_terms: D000818:Animals; D001216:Asparagine / Q000737:chemistry; D002401:Cathepsin B / Q000737:chemistry; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry; D002645:Chickens; D018360:Crystallography, X-Ray; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics; D003546:Cysteine Endopeptidases; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: D015891:Cystatins; D005973:Glutamine; D001216:Asparagine; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D007930:Leucine