Three-Dimensional Solution Structure of the Reduced Form of Escherichia coli Thioredoxin Determined by Nuclear Magnetic Resonance Spectroscopy

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.