Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain

Abstract

~. IntroductionChicken cystatin, a small protein-type inhibitor of cysteine proteinases, represents a well studied member of the cystatin ,uperfamily (for reviews see [2,3]). It was shown to be a reversble tight-binding inhibitor of papain and papain-like enzymes 4Corresponding author. Fax: (49) (89) 5160 4735. E-mail: <auerswald@clinbio.med.uni-muenchen.de>tbbreviations. E-64, t.-3-carboxy-2,3-trans-epoxy-propionyl-leucyl~Lmido-(4-guanidino)butane; NH-Mec, 7-(4-methyl) coumaryl-amide; . [4-9]. Synthetic genes of chicken cystatin and variants of them have been expressed and characterized [10][11][12]. Crystal and solution structures of native and recombinant chicken cystatin are known [13,14] and a model of the interaction of chicken cystatin with papain has been proposed [7,13]. According to the so-called 'elephant-trunk model', the enzyme-inhibitor complex is formed mainly by hydrophobic interactions between chicken cystatin and the complementary active-site cleft of papain, whereby cystatin contributes the N-terminal 'trunk' (Leu7-Gly9), the first hairpin loop (Gln53-Gly57) and the second hairpin loop (Pro]°3-Leul°5). The validity of this model was confirmed by elucidation of the structure of the complex formed between S-carboxymethylated papain and stefin B [15] as well as by kinetic data obtained for the interactions of native and/or modified cystatins with different cysteine proteinases [7,8,12,16].Detailed analyses of the N-terminal segments of chicken cystatin and human cystatin C indicate a substrate-like interaction of this region with residues of the subsites $3 and $2 of the proteinase. Especially the amino acid residues preceding the conserved Gly 9 of chicken cystatin or Gly ~1 of human cystatin C have distinct but different effects on the binding to papain, actinidin, ficin, cathepsin B or cathepsin L [17 20].The contribution of the first hairpin loop of cystatins to complex formation has not yet been investigated in such detail. From studies with KVVAG-and QVTAG-mutants of cystatin A, Nikawa et al. [21] proposed that the first hairpin loop is not essential for cysteine proteinase inhibition. But the importance of this region is strongly suggested by (i) the structural data of the crystallized stefin B-papain complex [15], (ii) its conserved primary structure (QxVxG-region) [2] and (iii) inhibition properties measured with substitution variants of oryzacystatin [22] and chicken cystatin [12]. The latter data provide first evidence that amino acid residues of this region are partially responsible for the distinct affinities of cystatins for different cysteine proteinases. However, no data have been presented so far on variants of the first hairpin loop missing conserved amino acid residues.Little is known about the importance of the second hairpin loop for the stability of the molecule itself or its contribution to complex formation. Using spectroscopic methods, Lindahl and co-workers [6] demonstrated that Trpl°4 of chicken cystatin must be involved in the interaction with papain. These early data...

Temporary inhibition of papain by hairpin loop mutants of chicken cystatin Distorted binding of the loops results in cleavage of the Gly9‐Ala10 bond

“…time we have also observed temporary inhibition of the cysteine proteinases papain and cathepsin L during interaction with recombinant chicken cystatin variants bearing substitutions or deletions in one of the two hairpin loop binding regions [4]. In this report we present kinetic and structural data describing the temporary inhibition of papain and propose a putative model for its molecular mechanism.…”

“…Molecular cloning and expression of recombinant chicken cystatin variants have been described elsewere [4,[7][8][9]. Human cathepsin B (EC 3.4.22.1) and human cathepsin L (EC 3.4.22.15) were purchased from Medor (Herrsching) or Calbiochem (Bad Soden/Ts.)…”

“…Rate constants and Ki values were corrected for competition with the substrate. Active concentrations of the inhibitor variants were determined from inhibition experiments under equilibrium or incomplete titration conditions performed with defined concentrations of papain (titrated with E-64) or cathepsin L (titrated with standardized chicken cystatin) as described [4].…”

“…Kia and K~b were obtained from the v~a/v0 and Vib/vo ratios (see Fig. 1) as described [4]. If korr was known (see section 3.2), kon was calculated as kon = korr/Ki, else the value of ko.…”

“…The preparation and characterization of hairpin loop variants of recombinant chicken cystatin is described elsewhere [4]. The temporary nature of papain inhibition by these variants (Table 1) was discovered in experiments with enzyme concentrations above 0.5 nM.…”