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 sitedirected 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 300-fold, >10-fold and 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by 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 aminoacid 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, 45% of the total binding energy, for inhibition of cathepsin B.Keywords: cathepsins; cystatin; cysteine proteases; papain; second binding loop.Cystatins are effective protein inhibitors of cysteine proteases of the papain superfamily (reviewed in [1][2][3][4]). Found both intracellularly and extracellularly, they are believed to control the activity of normal endogenous proteases, as well as to protect organisms from the harmful activity of exogenous cysteine proteases [1,[4][5][6][7][8][9][10][11]. They are generally classified into three families according to their size and the presence of internal disulfide bonds. Cystatins of family 1, also called stefins, are small nonglycosylated proteins 11-12 kDa in size without disulfide bonds. Family 2 cystatins are somewhat larger, 12-14 kDa, with a structure stabilized by two disulfide bonds. Kininogens, representing the third family, are glycosylated proteins of about 50-90 kDa.The single polypeptide chain of a kininogen contains three domains resembling family 2 cystatins.Cystatins competitively inhibit the activity of papainlike cysteine proteases by binding to the active site of the latter and forming a tight, reversible protein-protein complex. A model of the inhibition was initially proposed from computer docking experiments based on the X-ray structures of papain and chicken cystatin, a family 2 member [12]. This model was later substantiated by the X-ray structure of a complex of the family 1 cystatin, human cystatin B (stefin B), with papain [13], the only structure of a cystatin-protease complex determined so far. The N-terminal segment and two hairpin loops of the cys...