Solution Structure and Dynamics of the Human−Escherichia coli Thioredoxin Chimera: Insights into Thermodynamic Stability,

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Abstract
We have determined the high-resolution solution structure of the oxidized form of a chimeric human and Escherichia coli thioredoxin (TRXHE) by NMR. The overall structure is well-defined with a rms difference for the backbone atoms of 0.27 ± 0.06 Å. The topology of the protein is identical to those of the human and E. coli parent proteins, consisting of a central five-stranded β-sheet surrounded by four α-helices. Analysis of the interfaces between the two domains derived from the human and E. coli sequences reveals that the general hydrophobic packing is unaltered and only subtle changes in the details of side chain interactions are observed. The packing of helix α4 with helix α2 across the hybrid interface is less optimal than in the parent molecules, and electrostatic interactions between polar side chains are missing. In particular, lysine−glutamate salt bridges between residues on helices α2 and α4, which were observed in both human and E. coli proteins, are not present in the chimeric protein. The origin of the known reduced thermodynamic stability of TRXHE was probed by mutagenesis on the basis of these structural findings. Two mutants of TRXHE, S44D and S44E, were created, and their thermal and chemical stabilities were examined. Improved stability toward chaotropic agents was observed for both mutants, but no increase in the denaturation temperature was seen compared to that of TRXHE. In addition to the structural analysis, the backbone dynamics of TRXHE were investigated by 15N NMR relaxation measurements. Analysis using the model free approach reveals that the protein is fairly rigid with an average S2 of 0.88. Increased mobility is primarily present in two external loop regions comprising residues 72−74 and 92−94 that contain glycine and proline residues.