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 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...

Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers

“…The leucine residue at position 73 mediates part of the interaction between SteA with its target proteases. 13,19 There is considerable sequence variability in the cystatin family around this residue (Figure 1(a)), suggesting that this region does not contribute either to the folding of the protein, or to interactions with partners. Various members of the cystatin family have insertions close Figure 1.…”

Grafting of Features of Cystatins C or B into the N-Terminal Region or Second Binding Loop of Cystatin A (Stefin A) Substantially Enhances Inhibition of Cysteine Proteinases

“…Expression vectors for human cystatin A variants with a V47I mutation in the first binding loop or G75H or G75W mutations in the second binding loop ( Figure 1) were obtained by a two-step, PCR-based site-directed mutagenesis procedure with the wild-type vector described above as template, essentially as in previous work (17,33). The mutations were introduced by the mutagenic primers 5′-GAAGCTGT-GCAGTATAAAACTCAAATTGTTGCTGGAAC (upstream) and 5′-CTTAATGTAGTAATTTGTTCCAGCAACAAT-TTGAGTTTTATAC (downstream) for V47I-cystatin A; 5′-CTTGAAAGTATTCAAAAGTCTTCCCCATCAAAA-1 Abbreviations: app, subscript denoting an apparent equilibrium or rate constant determined in the presence of an enzyme substrate; C29A-cathepsin B, human cathepsin B variant in which Cys29 is replaced by Ala; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate; DTT, dithiothreitol; G75H-and G75W-cystatin A, human cystatin A variants in which Gly75 is replaced by His and Trp, respectively; H110A/C29A-cathepsin B, C29A-cathepsin B variant in which H110 is replaced by Ala; His-tag, 10 consecutive histidine residues fused to an expressed protein; k ass, bimolecular association rate constant; Kd, dissociation equilibrium constant; kdiss, dissociation rate constant; Ki, inhibition constant; kobs, observed pseudo-first-order rate constant; Mes, 4-morpholineethanesulfonic acid; N(1-10)CC-and N(8-10)CC-cystatin A, human cystatin A variants in which the three initial amino-terminal amino acid residues are replaced by residues 1-10 and 8-10 of human cystatin C, respectively; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; V47I-cystatin A, human cystatin A variant in which Val47 is replaced by Ile.…”

“…The X-ray structure of a complex between cystatin B and a cysteine proteinase, papain (7), shows that the three binding regions establish a number of predominantly hydrophobic contacts but also polar interactions with the proteinase. Moreover, site-directed mutagenesis has demonstrated the importance of several individual residues in these regions of different cystatins for the interaction (9)(10)(11)(12)(13)(14)(15)(16)(17)(18).…”

Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant

“…Stefin A is a small (98 amino acid) monomeric protein inhibitor of the cystatin family I (stefins) that inhibits cysteine proteases of the cathepsin family (Turk et al ., 1986). It interacts with its partner proteins, cathepsins B, C, H, L and S (Brzin et al ., 1984; Green et al ., 1984; Abrahamson et al ., 1986) using three sites, with key contacts made by glycine at position 4, valine at position 48 and lysine at position 73 (Bode et al ., 1988; Stubbs et al ., 1990; Martin et al ., 1994, 1995; Tate et al ., 1995; Pavlova and Björk, 2002; Jenko et al ., 2003). Biological neutrality was achieved through a combination of two rational mutations [G4W to abolish interaction with cathepsins (Estrada et al ., 1998, 1999) and V48D to abolish interaction with cathepsins and reduce dimer formation through domain swapping (Japelj et al ., 2004)] and a third mutation to introduce a unique Rsr II restriction site at codons 71–73, which becomes the insertion site for oligonucleotides encoding short peptides.…”

“…Biological neutrality was achieved through a combination of two rational mutations [G4W to abolish interaction with cathepsins (Estrada et al ., 1998, 1999) and V48D to abolish interaction with cathepsins and reduce dimer formation through domain swapping (Japelj et al ., 2004)] and a third mutation to introduce a unique Rsr II restriction site at codons 71–73, which becomes the insertion site for oligonucleotides encoding short peptides. The use of this site allowed us to introduce peptides into the longest loop found in SteA, known as loop 2 (Bode et al ., 1988; Stubbs et al ., 1990; Martin et al ., 1994, 1995; Tate et al ., 1995; Pavlova and Björk, 2002). The engineered protein was designated STM, for stefin A triple mutant (Woodman et al ., 2005).…”