Mapping the serpin-proteinase complex using single cysteine variants of alpha1-proteinase inhibitor Pittsburgh.
To probe the covalent serpin-proteinase complex, we used wild-type and 4 new single cysteine variants (T85C, S121C, D159C, and D298C) of alpha1-proteinase inhibitor Pittsburgh. Cysteines in each variant could be labeled both in native and proteinase-complexed alpha1-proteinase inhibitors. Pre-reaction with 7-nitrobenz-2-oxa-1, 3-diazole-chloride or fluorescein prevented complex formation only with the D298C variant. Label at Cys121 greatly increased the stoichiometry of inhibition for thrombin and gave an emission spectrum that discriminated between native, cleaved, and proteinase-complexed serpin and between complexes with trypsin and thrombin, whereas fluorophore at residue 159 on helix F was almost insensitive to complex formation. Fluorescence resonance energy transfer measurements for covalent and non-covalent complexes were consistent with a location of the proteinase at the end of the serpin distal from the original location of the reactive center loop. Taken together, these findings are consistent with a serpin-proteinase complex in which the reactive center loop is fully inserted into beta-sheet A, and the proteinase is at the far end of the serpin from its initial site of docking with the reactive center loop close to, but not obscuring, residue 121.
Stratikos
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Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, Illinois 60612-4316, USA.
Gettins
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PG
D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008956:Models, Chemical; D008958:Models, Molecular; D010449:Peptide Mapping; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; D015843:Serpins; D000515:alpha 1-Antitrypsin; C038915:alpha 1-antitrypsin Pittsburgh; D012697:Serine Endopeptidases; D003545:Cysteine