Structural Plasticity of the Thioredoxin Recognition Site of Yeast Methionine S-Sulfoxide Reductase Mxr1

Xiao-Xiao Ma,Peng-Chao Guo,Wei-Wei Shi,Ming Luo,Xiao-Feng Tan,Yuxing Chen,Cong-Zhao Zhou

doi:10.1074/jbc.m110.205161

Abstract

The methionine S-sulfoxide reductase MsrA catalyzes the reduction of methionine sulfoxide, a ubiquitous reaction depending on the thioredoxin system. To investigate interactions between MsrA and thioredoxin (Trx), we determined the crystal structures of yeast MsrA/Mxr1 in their reduced, oxidized, and Trx2-complexed forms, at 2.03, 1.90, and 2.70 Å, respectively. Comparative structure analysis revealed significant conformational changes of the three loops, which form a plastic "cushion" to harbor the electron donor Trx2. The flexible C-terminal loop enabled Mxr1 to access the methionine sulfoxide on various protein substrates. Moreover, the plasticity of the Trx binding site on Mxr1 provides structural insights into the recognition of diverse substrates by a universal catalytic motif of Trx.

Highlights

Methionine is one of the most sensitive amino acid residues subject to oxidation
Previous report on bovine molecular replacement using the central domain (MsrA) has shown that mutation of the cysteine corresponding to Mxr1Cys68 did not disturb the methionine sulfoxide (Met-SO) reduction [40]
Structural Plasticity of MsrA and MsrB—Oxidation of the methionine would lead to various deleterious effects, ranging from decline of the enzymatic activity to inactivation of the proteins

Summary

EXPERIMENTAL PROCEDURES

Construction, Expression, and Purification of Mxr and Mutants—The MXR1 gene was amplified by PCR from genomic DNA of Saccharomyces cerevisiae S288C. Crystallization, Data Collection, and Processing—Crystals of the reduced, oxidized, and Trx2-complexed Mxr were grown by hanging-drop vapor diffusion at 16 °C, with the initial condition of mixing 1 ␮l of protein sample at 25 mg/ml with an equal volume of the reservoir solution (for the reduced form, 0.2 M ammonium acetate, 0.1 M sodium acetate, pH 4.6, 30% polyethylene glycol 4,000, 10 mM dithiothreitol; for the oxidized form, 0.5 M ammonium sulfate, 0.1 M sodium citrate, pH 5.6, 1.0 M lithium sulfate; for the Trx-complexed form, 0.2 M trimethylamine N-oxide, 0.1 M Tris-HCl, pH 8.5, 20% polyethylene glycol 2,000 monomethyl ether) The crystals of the reduced and Trx2-complexed forms appeared in 3 days and reached the maximum size in 1 week, whereas the oxidized form appeared in 4 months. All structure figures were prepared with PyMOL [39]

RESULTS

PDB code

DISCUSSION