Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer

被引:24
作者
Thompson, LC
Walters, J
Burke, J
Parsons, JF
Armstrong, RN
Dirr, HW
机构
[1] Vanderbilt Univ, Sch Med, Ctr Mol Toxicol, Dept Biochem, Nashville, TN 37232 USA
[2] Vanderbilt Univ, Sch Med, Ctr Mol Toxicol, Dept Chem, Nashville, TN 37232 USA
[3] Univ Witwatersrand, Sch Mol & Cell Biol, Prot Struct Funct Res Unit, ZA-2050 Johannesburg, South Africa
[4] Maryland Biotechnol Inst, Ctr Adv Res Biotechnol, Rockville, MD 20850 USA
[5] Natl Inst Stand & Technol, Rockville, MD 20850 USA
基金
英国惠康基金;
关键词
D O I
10.1021/bi0519506
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry, 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A Mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double Mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A Mutants indicate that a reorganization of the loop structure between helix alpha 2 and strand beta 3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha 4 and alpha 5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A Mutant Suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.
引用
收藏
页码:2267 / 2273
页数:7
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