The three-dimensional solution structure of reduced (dithiol) thioredoxin from Escherichia coli has been determined with distance and dihedral angle constraints obtained from 1H NMR spectroscopy. Reduced thioredoxin has a well-defined global fold consisting of a central five-strand β-sheet and three long helices. The β-strands are packed in the sheet in the order β1β3β2β4β5, with β1,β3,and β2 parallel and β2, β4, and β5 arranged in an antiparallel fashion. Two of the helices connect strands of the β-sheet: α1 between β1, and β2 and α2 between β2 and β3. Strands β4 and β5 are connected by a short loop that contains a β-bulge. Strands β3 and β4 are connected by a long loop that contains a series of turn-like or 310 helical structures. The active site Cys-Gly-Pro-Cys sequence forms a protruding loop between strand β2 and helix α2. The structure is very similar overall to that of oxidized (disulfide) thioredoxin obtained from X-ray crystal structure analysis but differs in the local conformation of the active site loop. The distance between the sulfurs of Cys 32 and Cys 35 increases from 2.05 Å in the disulfide bridge to 6.8 ± 0.6 Å in the dithiol of reduced thioredoxin, as a result of a rotation of the side chain of Cys 35 and a significant change in the position of Pro 34. This conformational change has important implications for the mechanism of thioredoxin as a protein disulfide oxidoreductase.
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