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

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. colithioredoxin for chloroplast fructose-1,6-bisphosphatase (A 0.5 = 9, 7, and 3 μm, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A 0.5 = 33 μm), though still lower than that of thioredoxin-f (A 0.5 = 0.9 μm). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A 0.5for all bacterial variants to a limiting value of ∼9 μmbut 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. 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. colithioredoxin for chloroplast fructose-1,6-bisphosphatase (A 0.5 = 9, 7, and 3 μm, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A 0.5 = 33 μm), though still lower than that of thioredoxin-f (A 0.5 = 0.9 μm). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A 0.5for all bacterial variants to a limiting value of ∼9 μmbut 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. The superfamily of Trx 1The abbreviations used are: Trx(s), thioredoxin(s); CFBPase, chloroplast fructose-1,6-bisphosphatase; di-FTC-insulin, difluoresceinthiocarbamyl-insulin; IPTG, isopropyl-β-d-thiogalactopyranoside; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; Tricine,N-tris(hydroxymethyl)methylglycine. comprises small proteins (∼12 kDa) whose distinctive feature is the amino acid sequence (-W-C-G-P-C-) functional in thiol/disulfide exchange with other proteins (1Eklund H. Gleason F.K. Holmgren A. Proteins Struct. Funct. Genet. 1991; 11: 13-28Crossref PubMed Scopus (329) Google Scholar). Their widespread occurrence and structural stability are matched by a range of biological properties from metabolic regulation (2Wolosiuk R.A. Ballicora M.A. Hagelin K. FASEB J. 1993; 7: 622-637Crossref PubMed Scopus (60) Google Scholar) to virus replication (3Huber H.E. Russel M. Model P. Richardson C.C. 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To we used a small highly (pI = and protein in of the protein-disulfide reductase of Trx was in the presence of NADPH and NADP-thioredoxin reductase napin the rate of disulfide reduction was and of NADPH for the wild E30K, L94K, and E30K/L94K The of positive charges on the of Trx a in the rate of napin A of thiol/disulfide the of reducing in the photosynthetic with the of the of the of two proteins to the active site of ferredoxin-Trx reductase to the disulfide of the of the oxidized and the protein to sulfhydryl groups of the reduced In the of the is to structural that the affinity of reduced Trx for that to the specific of chloroplast located at residues that not in the active site of all amino to in the of charges, a of and Schürmann P. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). the other for the activation of in a by high density of negative charges, Y. Y. 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