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.
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.
Pavlova
Alona
A
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Sweden.
Björk
Ingemar
I
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
D015891:Cystatins; D005973:Glutamine; D001216:Asparagine; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D007930:Leucine