5584 B I O C H E M I S T R Y Smith, L. (1955) in Methods in Enzymology ZZ (Colowick, S . P., & Kaplan, N. O., Eds.) p 732, Academic Press, New York. Stahl, P. D., & Touster, 0. (1971) J. Biol. Chem. 246, 5398. Tauber, A. I., & Babior, B. M. (1977) J. Clin. Invest. 60, 374. Tauber, A. I., & Babior, B. M. (1978) Photochem. Photobiol. 28, 701.
SLABY AND HOLMGREN
Tauber, A. I., Gabig, T. G., & Babior, B. M. (1 979a) Blood 53, 666. Tauber, A. I., Goetzl, E. J., & Babior, B. M. (1979b) Znflammation ( N .Y.) 3, 26 1. Tsan, M.-F. (1978) RES, J. Reticuloendothel. SOC.23, 205. West, B. C., Rosenthal, A. S., Gelb, N. A., & Kimball, H. R. (1974) Am. J . Pathol. 77, 41.
Structure and Enzymatic Functions of Thioredoxin Refolded by Complementation of Two Tryptic Peptide Fragmentst Ivan Slabyt and Arne Holmgren*
ABSTRACT: The physicochemical and catalytic properties of thioredoxin-T’ are described. This complemented protein structure consists of a 1:l complex between the inactive fragments thioredoxin-T-( 1-73) and thioredoxin-T-(74-108). These are generated by selective trypsin cleavage at Arg-73 in lysine-modified and denatured Escherichia coli thioredoxin. Thioredoxin-T’ was a slowly formed but stable complex with an apparent K D below lo-* M. The tryptophan fluorescence spectrum and the CD spectrum were very similar to those of native thioredoxin; some conformational differences were detected by gel chromatography and radioimmunoassay.
Thioredoxin-T’-S2 was a substrate for NADPH and thioredoxin reductase and had l-2% of the activity of native thioredoxin. This low relative activity was the result of a major increase in the K,,, value. Thioredoxin-(SH), was a hydrogen donor for E . coli ribonucleotide reductase with about 3% relative activity. These results for thioredoxin-T’ are correlated with the known three-dimensional structure of thioredoxin. The microenvironment around Arg-73 that is close to the active disulfide appears to be of critical importance for the interactions of thioredoxin with thioredoxin reductase and ribonucleotide reductase.
zioredoxin-S,1 contains an intramolecular cystine disulfide A-Sbridge (-Cys,,-Gly-Pro - C Y S ~ ~as- )the catalytically important group (Holmgren, 1968). This disulfide is reduced to a dithiol with NADPH in a reaction catalyzed by the specific flavoprotein thioredoxin reductase [for a review, see Holmgren (1980)l. Thioredoxin-(SH), is an efficient disulfide reductase and is reoxidized in reactions such as those shown in Figure 1. The assays for thioredoxin activity are thus based on the cyclic oxidoreduction of thioredoxin in the presence of NADPH and thioredoxin reductase and a disulfide acceptor. A novel and so far largely unknown function of Escherichia coli thioredoxin is to be the host-coded essential subunit of phage T7 DNA polymerase (Mark & Richardson, 1976), required for virus DNA replication in vivo and in vitro. Previous studies of the structure and function of thioredoxin have been aimed at an understanding of its molecular mechanism of action. Thus, the complete primary structure of the 108 amino acid residues of thioredoxin-S2 from E . coli has been determined (Holmgren, 1968). The three-dimensional structure of thioredoxin-S, has been solved to 2.8-A resolution by X-ray crystallographic techniques (Holmgren et al., 1975). In addition, we previously identified two systems of peptide fragments that can be used to reconstitute thioredoxin-S2 by noncovalent complementation (Holmgren, 1972a; Slab9 & Holmgren, 1975). Cleavage of thioredoxin with cyanogen
bromide at the single methionine residue (Met-37) yields the two peptide fragments thioredoxin-C-( 1-37) and thioredoxin-C-(38-108) that upon mixing form thioredoxin-C’. Selective cleavage by trypsin of thioredoxin, where all the lysines have been blocked by citraconic anhydride, splits the molecule at the single arginine residue (Arg-73). The resulting peptide fragments, after removal of citraconyl groups by mild acid hydrolysis, thioredoxin-T-( 1-73) and thioredoxin-T(74-108), interact specifically to give thioredoxin-T’ (Slab9 & Holmgren, 1975). Thioredoxin-T’ and thioredoxin-C’ both give full immunoprecipitation activity with rabbit antibodies against native thioredoxin and have a low but significant enzymatic activity with thioredoxin reductase (Slab9 & Holmgren, 1975). The component peptides of these complexes are enzymatically inactive. However, thioredoxin-T-(74-108) and also thioredoxin-C-(38-108) were strong inhibitors of the precipitation reaction of native thioredoxin-S2 with its antibodies. This demonstrated that one of the major antigenic determinants of thioredoxin is contained in the thioredoxin-T-(74-108) amino acid sequence. Furthermore, the results show that this COOH-terminal fragment has the capacity of nucleated folding into a structure similar to that which it normally occupies in native thioredoxin. This conclusion has been strongly supported by the three-dimensional structure of thioredoxin-S2
From the Department of Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden. Received June 27, 1979; revised manuscript receiued September 26, 1979. This investigation was supported by grants from the Swedish Medical Research Council Projects 13X-3529 and 13P-4292, Magnus Bergvalls Stiftelse, and by a short-term EMBO fellowship to IS. *Present address: Department of Medical Chemistry, Charles University, Karlovarska 48, Plzen, Czechoslovakia.
Abbreviations used: thioredoxin-S2 (T-S2) and thiored~xin-(SH)~ [T-(SH)2],the oxidized and reduced forms of thioredoxin, respectively; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid). Peptide fragments have been designated by an adoption of the rules of the IUPAC-IUB Commission on Biochemical Nomenclature. Fragments obtained after CNBr cleavage are denoted by C and after selective cleavage with trypsin are denoted by T. The reconstituted noncovalent complexes are denoted thioredoxin-C’ and thioredoxin-T’ (Slab5 & Holmgren, 1975).
0006-2960/79/0418-5584$01 .OO/O
’
0 1979 American Chemical Society
CHARACTERIZATION OF THIOREDOXIN-T’
METHOD 1
R -S-S-R
spontaneous
>
ZR-SH
3
tnsul in
METHOD 2
/s
’su‘in-\i spontaneous
NADP+’
Thioredoxin reductase
’ NADPH
S , thioredoxin-T-(74-108)
0.34 0.10 0.29 0.22 0.18
0.37
A column of Sephadex G 5 0 (0.9 X 140 c m ) equilibrated with KAV values (Laurent 0.5% ammonium bicarbonate was used. & Killander, 1964) were calculated according to the equation K A V , = ( V c - V , ) / ( V , - V , ) where Vc is elution volume of the protein and VT is the total volume of the column. Vo for this Sephadex G 5 0 column was determined to be 33% of VT.
to dissociate in the chromatographic procedures, showing that it is a stable complex. Characterization of Thioredoxin-T' and the Fragments by Gel Chromatography. Gel chromatography on a calibrated column of Sephadex G-50 was used to measure elution volumes and to calculate KAvvalues (Laurent & Killander, 1964) for thioredoxin-S2,citraconylated thioredoxin, thioredoxin-T', and the peptide fragments (Table I). Citraconylated thioredoxin, a totally inactive highly acidic molecule (Slab? & Holmgren, 1975), had the lowest KAV value, indicating a random-coil structure with a Stokes radius larger than that of native thioredoxin-S2 (Figure 2B). Thioredoxin-S2 is known from the three-dimensional structure (Holmgren et al., 1975) to be an essentially spherical molecule with overall dimensions of 25 X 34 X 35 A. Thioredoxin-T-(1-73) had a KAv value which indicated that it is mainly a random coil. The somewhat high value for thioredoxin-T-(74-108) may reflect a lower molecular weight and a compact size consistent with some folding. The citraconylated peptide fragments had essentially the same KAv values on Sephadex G-50 chromatography as the deblocked fragments. The formation of thioredoxin-T'-Sz
CHARACTERIZATION OF THIOREDOXIN-T'
nbn7
--
- . ..
CATHODE
E
FIGURE 3: Polyacrylamide gel electrophoresis of thioredoxin-T'. (A) IO fig of each of (1) native thioredoxin. (2) thioredoxin-T', (3) mixed
thioredoxin and thioredoxin-T', and (4) mixed thioredoxin and thioredoxin-T-(l-73) was run on the polyacrylamide gels at pH 8.9. (B) 10 pg each of (1) native thioredoxin, (2) thioredoxin-T-(l-73), (3) thioredoxin-T', and (4) mixed native thioredoxin and thioredoxin-T' was run on gels containing 7 M urea at pH 8.9. The small peptide thioredoxin-T-(74-108) was not stained. was accompanied by a folding to a thioredoxin-like structure as seen from the KAv values of Table I. However, in all experiments the KAv value of thioredoxin-T'-S, was seen to be significantly lower than that of thioredoxin-S2. This is consistent with a more flexible structure of thioredoxin-T', resulting in a larger effective Stokes radius. Characterization by Polyacrylamide Gel Electrophoresis. This was performed in order to compare the behavior of the peptide fragments, thioredoxin-T', and thioredoxin under native and denaturing conditions (Figure 3). Peptide-T-(74-108) could not be visualized in the gels since it was not stained despite the use of trichloroacetic acid in the staining procedure. Under nondenaturing conditions (pH 8.9). thioredoxin-S, showed a high anodical mobility consistent with its acidic isoelectric point of 4.5 (Holmgren, 1968). Thioredoxin-T(I-73), which has a lower molecular weight and is even more acidic than thioredoxin (Figure 4). showed a lower mobility. This indicates that the fragment has a larger effective Stokes radius than native thioredoxin, corresponding to a random-coil structure. Furthermore, the fragment had the same mobility a t pH 8.9 in 7 M urea, strongly suggesting that it was a random coil under native conditions and not a dimer. In contrast, thioredoxin had a markedly lower mobility in 7 M urea a t pH 8.9, indicating the unfolding of the molecule. Indeed, as seen in Figure 38, thioredoxin in 7 M urea showed a trailing forward, suggesting a finite equilibrium between a folded and unfolded form of the protein. Thioredoxin-S, is known to be quite resistant to unfolding in urea (Holmgren, 1972b). Thioredoxin-T' moved faster than thioredoxin at pH 8.9 in native gels (Figure 3A). This behavior is expected since a t pH 8.9 the COOH group of Arg-73 carries a negative charge not present in native thioredoxin. The preparations of thioredoxin-T' contained also varying amounts of a second band with even faster mobility. This probably arises from partial deamidation of Asn-83 under the acidic conditions of preparation of the peptide fragments (Holmgren, 1968;
FIGURE 4 The amino acid sequence of thioredoxin-S2from E. coli including the semndary structure elements, u helices, &pleated sheets, and reverse turns (R) as determined from the three-dimensional structure (Holmgren et al., 1975). The specific cleavage point at Arg-73 by trypsin (T) is indicated by an arrow. The cleavage at Met-37 by CNBr is also indicated by an arrow
Table 11: Enzymatic Activity of Thioredoxin-T' and Thioredoxin with Thioredoxin Reductase Measured bv Reduction of DTNB enzymatic act.' thiorcdoxin-T' (M) AA,,, X 1 X 10-6
4 x 10.6 8 X 10.'
2 x 10-5 4 x lo-'
min-'
0.015 0.060 0.165 0.320 0.560
% of thiorcdwin
1.07 1.07 I .47 1.14 1.00
Activity was determined by method I (see Experimental Procedure). The concentration of thioredoxin reductase \vas 1.0 x IO-' M. Thioredoxin showed an activity of AA,,, X min-' of 0.140 at 1.0 X IO-' M and the AA,,, X min-' w a s proportional to thiorcdoxin concentration in the range from 0 to 1.0 AA,,, x min-'. A controlsample of citraconylated thioredoxin that was subjected to the same 50% acetic acid chromatography as thc peptide fragments showed 52% of the activity of native
thioredoxin. Holmgren & Slaby, 1979). The gels show no trace of thioredoxin in the thioredoxin-T' preparations. Furthermore, the band of thioredoxin-T' was sharp, and no band corresponding to thioredoxin-T-( 1-73) was seen in the thioredoxin-T' preparation. This is consistent with a strong complex of thioredoxin-T' without any detectable dissociation. In contrast, thioredoxin-T' in 7 M urea showed only one band corresponding to thioredoxin-T-(1-73). The results demonstrate that thioredoxin-T' is a noncovalent complex and that it is not stable under the denaturing conditions of 7 M urea. Enzymatic Activity. The two peptide fragments thioredoxin-T-(l-73) and thioredoxin-T-(74-108) of thioredoxin-T' were completely inactive as substrates for thioredoxin reductase (I X IO-' M) at l X M concentration (Figure 5). The purified complex thioredoxin-T' had activity with thioredoxin reductase as measured by the reduction of DTNB with excess thioredoxin reductase (Table 11). Thioredoxin-T' showed around l% of the activity of native thioredoxin in the range from l X IO" to 40 X 10" M. There
5588
B 1OC H E M I S T R Y
SLABY AND HOLMGREN
Table IV: Activity of Thioredoxin-T‘-(SH), as Hydrogen Donor for E. coli Ribonucleotide Reductase”
Q7 hydrogen donor
concn (M)
thioredoxin-(SH), thioredoxin-T-( 1-73) thioredoxin-T-(7 4- 108) thioredoxin-T’-(SH), thioredoxin-T-(l-73) plus thioredoxin-( SH),
4 x 10-l 1.0 x 1 0 - ~ 1.0 x lor5 1.1 x 10 ’ 1.0 x l o - ’ plus 4 x lo-’
0.6 0.5
G I-
d
0.L
W
7.1
