The crystal structure of chicken egg white cystatin has been solved by X‐ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X‐ray data between 6 and 2.0A. The molecule consists mainly of a straight five‐turn alpha‐helix, a five‐stranded antiparallel beta‐pleated sheet which is twisted and wrapped around the alpha‐helix and an appending segment of partially alpha‐helical geometry. The ‘highly conserved’ region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta‐hairpin loop which on opposite sides is flanked by the amino‐terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I ‐ Trp104I. These loops and the amino‐terminal Gly9I ‐ Ala10I form a wedge‐shaped ‘edge’ which is quite complementary to the ‘active site cleft’ of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino‐terminal segment Gly9I ‐ Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin‐like inhibitors seems to be fundamentally different from the ‘standard mechanism’ defined for serine proteinases and most of their protein inhibitors.

N-terminally truncated forms of chicken egg white cystatin and its cyanogen bromide fragments were isolated and assayed for inhibition of papain. Truncated forms beginning with Gly-9 and Ala-10 had a 5000-fold lower affinity for papain than the two isoelectric forms (pi=6.5 and 5.6) of the full-length inhibitor (K~--6 pM and 7 pM) or a truncated form beginning with Leu-7 (/~ = 6 pM), indicating the outstanding importance of one or two residues preceding conserved Gly-9 for binding. A weak inhibition of papain (K~ = 900 nM) was exhibited by the intermediate cyanogen bromide fragment (residues 30-89) containing the chicken cystatin QLVSG variation of the QWAG segment which is conserved in almost all members of the cystatin superfamily. The obtained affinity data provide independent evidence for the validity of the proposed docking model of a chicken cystatin-papain complex [( ) EMBO J. 7, 2593[( -2599.

A synthetic gene coding for the E. coli proteins) and mostly deposited as inhuman intracellular cysteine proteinase inhibitor, elusion bodies. The unfolded fusion protein was stefin B, was constructed from 13 chemically partially purified in the presence of urea. After synthesized oligonucleotides according to the refolding, approx. 6% of the protein was inmethod of Khorana. The gene was inserted into hibitorily active against papain, human cathepsin the plasmid vector pTZ, amplified and se-H and B. Des[Metl' 2 ]stefin B was released by quenced. For expression, a temperature-induecyanogen bromide cleavage of the fusion protein ible system producing fusion proteins was used. and identified by N-terminal amino-acid seWith the vector pEx31A containing the synthetquence analysis. The non-separated cleavage ic cystatin B gene, E. coli strain 537 produced a products were also inhibitorily active after refusion protein of the N-terminal part of bacterio-folding. The estimated inhibition constants for phage MS-2 polymerase and [Met~2Gly~1 ]stefin the fusion protein and its cleavage products B. Lysates of the induced bacteria were inhibiwere similar to those reported for natural torily active against papain. The fusion protein stefin B. was expressed in high yield (about 20%

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