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- Kozak, M, et al.
(författare)
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Expression of a selenomethionine derivative and preliminary crystallographic studies of human cystatin C
- 1999
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 55:11, s. 1939-1942
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain-like mammalian proteases, has been produced in its full-length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met→Se-Met substitution confirmed by mass spectrometry, amino-acid analysis and X-ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N-terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full-length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.
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2. |
- Alvarez Fernandez, Marcia, et al.
(författare)
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Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site
- 1999
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 274:27, s. 19195-19203
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The Ki value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with Ki values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low Mr kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (Ki 0.0016 nM) and relatively weak (Ki 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the alpha-helix and the first strand of the main beta-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (Ki 1.5 nM) but >>5,000-fold lower affinity for legumain (Ki >>1,000 nM) than wild-type cystatin C.
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