Role of Electrostatic Interactions on the Affinity of Thioredoxin for Target Proteins

Abstract

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1,6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/ disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/ L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A 0.5 ‫؍‬ 9, 7, and 3 M, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A 0.5 ‫؍‬ 33 M), though still lower than that of thioredoxin-f (A 0.5 ‫؍‬ 0.9 M). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A 0.5 for all bacterial variants to a limiting value of ϳ9 M but also increases the A 0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI ‫؍‬ 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI ‫؍‬ 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.

Thioredoxin-dependent Enzymatic Activation of Mercaptopyruvate Sulfurtransferase

“…After C154S, C263S, and C154S/C263S MSTs were incubated on ice for 3 days, each activity was decreased via oxidation of each catalytic site Cys 247 , and DTT and reduced Trx well restored the activity. 4 The results indicate that C154S, 4 N. Nagahara, unpublished data. A balance of reduced Trx and dissolved oxygen regulates the monomerdimer equilibrium in this in vitro study.…”

“…These findings are consistent with the finding that rat recombinant MST had a prominently broad band in a native PAGE. 4 Trx also regulates the activity of ␣-glucan, water dikinase in a higher plant, and Trx f most effectively affects the enzyme activity via reduction of the intrasubunit disulfide bond among Trx f , Trx mf , and DTT (34). The specificity of Trx f for this enzyme is determined by a consensus sequence "CFATC" contained in the active site.…”

“…Reduced thioredoxin (Trx) 2 cleaves the disulfide bond, "turns on or off a redox switch," and facilitates protein function or activates an enzyme that results from possible conformational changes (1)(2)(3)(4)(5)(6)(7)(8). These disulfide bonds are generally "intrasubunit or intramolecular disulfide bonds."…”

NTRC links built-in thioredoxin to light and sucrose in regulating starch synthesis in chloroplasts and amyloplasts

“…4) but higher than the more potent interaction of fructose-1,6-bisphosphatase with Trx f (0.9 M; ref. 14). This property indicates that the Trx module forming part of NTRC has a comparable or even higher affinity for AGPB than free Trx.…”

Activation of the Potato Tuber ADP-glucose Pyrophosphorylase by Thioredoxin

“…Pase by spinach leaf thioredoxin f was 4.6 M, higher than the A 0.5 for the most potent interactions between thioredoxin and its specific enzyme targets like fructose-1,6-bisphosphatase and thioredoxin f (0.9 M) (22), but also much lower than nonspecific interactions between fructose-1,6-bisphosphatase and E. coli thioredoxin (33 M). Recently, it was shown that the main factor in the specificity of thioredoxins m, f, and bacterial thioredoxins is manifested by electrostatic interactions (22). When the interactions are shielded or abolished by mutagenesis, the A 0.5 tends to reach values of ϳ10 M in all cases.…”