Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A.
To determine the location of the proteinase in the covalent serpin-proteinase complex we prepared seven single-cysteine-containing variants of the Pittsburgh variant of the serpin alpha1-proteinase inhibitor, and we labeled each cysteine with the dansyl fluorophore. The dansyl probes were used to determine proximity of the proteinase trypsin in covalent and noncovalent complexes with the serpin, both by direct perturbation and by fluorescence energy transfer from tryptophans in trypsin to dansyl. Large direct effects on dansyl fluorophores were seen for only two positions in covalent complex and one position in noncovalent complex. Distances ranging from <14 A to 64 A were used to severely constrain possible structures for the complex. The structure consistent with both distance constraints and direct perturbations of the dansyl fluorophores placed the proteinase at the distal end of the serpin from the initial docking site. This position for the proteinase requires complete translocation of the proteinase from one end of the serpin to the other and full insertion of the reactive center loop into beta-sheet A to form the kinetically trapped complex. The consequent tight juxtapositioning of serpin and proteinase could explain how distortion of the proteinase active site can occur and hence how many combinations of serpin and proteinase can be inhibited by a common conformational change mechanism.[1]References
- Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A. Stratikos, E., Gettins, P.G. Proc. Natl. Acad. Sci. U.S.A. (1999) [Pubmed]
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