Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions

Abstract

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.

Structural contributions of Delta class glutathione transferase active-site residues to catalysis

“…It was striking that all four amino acid replacements resulted in decreased CDNB-binding affinity concomitant with positive co-operativity effects ( Table 2). The cooperativity for CDNB substrate was previously reported in a study of subunit interface residues of the same enzyme, adGSTD4-4 [43].…”

Structural Analysis of Cysteine S-Nitrosylation: A Modified Acid-Based Motif and the Emerging Role of Trans-Nitrosylation

“…The importance of their subunit interface region is well understood and fundamental to the function of these proteins [37]. Two types of interaction emerged as critical for GSTs dimerization: hydrophobic contacts (particularly in the Alpha, Mu and Pi classes) and electrostatic interactions driven by class-specific patterns of charged amino acids exposed in the contact region [38]. We modeled a rat GSTt dimer based on the crystal structure of the human protein (1LJR which could not be directly used due to significant differences with the rat protein, including Cys50Ser mutation).…”

Conserved Residues in the Subunit Interface of tau Glutathione S‐transferase Affect Catalytic and Structural Functions

“…It has a low fluorescence yield in polar environments, which is greatly enhanced on interaction with many proteins. Thus, ANS is often used to monitor the appearance/disappearance of hydrophobic patches or surfaces on the proteins that were undergoing structural changes (Schonbrunn et al 2000; Piromjitpong et al 2007). When ANS was bound to the enzymes, the fluorescence intensity was enhanced, accompanied by a blue shift in its fluorescence‐emission maximum from 520 nm (free ANS in phosphate‐buffered saline [PBS] buffer) to 500 nm for the wild type and mutant enzymes (Figure 5).…”

“…The dimeric quaternary structure is required for the formation of a fully functional active site, part of which is located near the subunit interface (Sayed et al 2000). In alpha, mu, pi and delta GSTs, interactions at the subunit interface play important roles in specifying molecular recognition between subunits and in stabilizing the dimeric structures (Stenberg et al 2000; Luo et al 2002; Alves et al 2006; Wongsantichon and Ketterman 2006; Piromjitpong et al 2007). To date, studies on plant tau GSTs have focused mainly on structure and function of glutathione‐binding site (G‐site) and hydrophobic binding site (H‐site) residues (Zeng et al 2005; Zeng and Wang 2005; Axarli et al 2010; Lo Piero et al 2010).…”