From the Medical Nobel Institute for Biochemistry, Karolinska Institute, Stockholm S-171 77, Sweden
Received for publication, March 5, 2003
, and in revised form, March 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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E. coli thioredoxin catalyzes thiol-disulfide exchange reactions via two redox-active vicinal thiols (C32GPC35). The enzyme can thus be in an oxidized (Trx-S2) or reduced (Trx-(SH)2) state. Trx-(SH)2 can reduce exposed disulfides in many proteins, among others ribonucleotide reductase, methionine sulfoxide reductase, and 3'-phosphoadenosine 5'-phosphosulfate reductase (4). The molecule has a central core formed by three parallel and two anti-parallel strands of pleated sheets, which are surrounded by four
helices (thioredoxin fold) (4). The active site thiols of thioredoxin are located on a protruding turn between
2 and
2 consisting of residues 2937. Loop residues glycine 33, 7576, and 9193 are adjacent to the active site with which they form a flat hydrophobic surface believed to be involved in interactions with other proteins (5, 6). Trp28, Trp31, and Pro34 are also participating in the contact area (7). Solution structures of Trx-S2 and Trx-(SH)2 are similar with most differences occurring in the conformation in and around the active site (8). The gene of thioredoxin (trxA) is not essential for E. coli, however, its presence is mandatory for the growth of phages M13, f1, and T7 (9). In the case of phage T7, thioredoxin is essential for the DNA replication of the phage (1, 10).
The crystal structure of T7 DNA polymerase complexed with primer-template revealed that thioredoxin binds to an extended mobile 71-residue loop located at the tip of the thumb of g5p (11). The active site cysteines (Cys32 and Cys35) and Arg73-Gly74-Ile75-Pro76 of thioredoxin are buried in the 71-amino acid loop. Only reduced thioredoxin can bind to g5p (12). How thioredoxin increases the processivity of g5p is not clearly understood. Incorporation of the thioredoxin binding area of g5p into the thumb of E. coli DNA polymerase I (Klenow fragment of DNA polymerase) also increased its processivity in a thioredoxin-dependent manner (13).
To investigate further the interaction between thioredoxin and g5p, we have used surface plasmon resonance (SPR) to determine in real-time the association (ka) and dissociation (kd) rate constants rates and the resulting equilibrium dissociation constant (KD). The effect of temperature on KD was used to calculate changes in Gibbs free energy, enthalpy, and entropy that occur when g5p interacts with thioredoxin.
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MATERIALS AND METHODS |
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Wild type thioredoxin was overexpressed in E. coli strain SK 3981 containing plasmid pBHK8 with the trxA gene inserted in pBR325 (13). All mutants were prepared previously (14, 15) by site-directed mutagenesis and were overexpressed in strain JF521 (trxA), a generous gift of J. Fuchs (Department of Biochemistry, Gortner laboratory, St. Paul, MN). Thioredoxins were purified as described before (17). g5p was purified from E. coli BH 215/pRS 101 (18) using an Affi-Gel-thioredoxin affinity chromatography method (19).
The following extinction coefficients at 280 nm were used: 13,700 M1·cm1 for wild type thioredoxin and mutants D26A, C35A, P34S, P40G, and Y49F; 8,200 M1·cm1 for thioredoxin W31A, W31H, and W28A; 10,400 M1·cm1 for thioredoxin W31Y; 8,300 M1·cm1 for thioredoxin W31F; 134,000 M1·cm1 for g5p.
Immobilization of ThioredoxinSurface plasmon resonance experiments were carried out using a Biacore 1000 instrument. In initial experiments, thioredoxin was immobilized directly on the dextran surface of a CM5 chip after its activation with 2x 40 µl of a 1:1 mixture of NHS (75 mg/ml) and EDC (11.5 mg/ml). Thioredoxin was then injected (4060 µl of 50100 µg/ml) in 10 mM sodium acetate buffer, pH 4.25, and remaining active groups were blocked with 40 µl of 1 M ethanolamine, pH 8.5. Immobilized thioredoxin was reduced (60 µl of 1 µg/ml of E. coli thioredoxin reductase and 8 mM NADPH in HBS) and treated with 2 x 10 µl of glycine buffer, pH 11.2, to remove any non-covalently bound protein. Extensive washes with HBS, 2.5 mM dithiothreitol (DTT) preceded g5p injections.
Our work mainly involved thioredoxins immobilized on a CM5 chip through 6-amino-n-hexanoic acid, a six-carbon spacer. The dextran surface was activated with a 1:1 mixture of NHS and EDC, 6-amino-n-hexanoic acid (60 µl of 1 M, pH 8.5) was injected over the activated surface, and unreacted activated groups were blocked by ethanolamine. The carboxylic acid group of immobilized 6-amino-n-hexanoic acid was activated (40 µl of 1:1 NHS-EDC), reduced thioredoxin species were injected (4060 µl of 50100 µg/ml in 10 mM sodium acetate buffer, pH 4.25), and remaining activated groups were blocked with ethanolamine. The chip was washed with glycine buffer (2 x 10 µl, pH 11.2) to remove any non-covalently bound protein and finally kept in HBS.
Throughout all immobilizations, the flow rate was kept at 5 µl/min except for the immobilization of 6-amino-n-hexanoic acid which was performed at 4 µl/min. Immobilization of thioredoxins was kept at 200800 resonance units.
Interaction of g5p with Trx-(SH)2The association of g5p and Trx-(SH)2 was followed at 25 °C by injecting 75 µl of 50250 nM g5p in HBS with 2.5 mM DTT. Injection of g5p was initiated 400 s after the beginning of sensorgram monitoring and estimations for ka took into consideration the 4001250 s part. Dissociation was monitored in HBS, 2.5 mM DTT (150 µl), 1250 s after the beginning of sensorgram monitoring. Estimations for kd were based on the data for 12502450 s. Any residual bound g5p was removed by 10 µl of glycine buffer, pH 11.2. To investigate the effect of temperature on dissociation constants, experiments were carried out at 20, 25, and 30 °C with a flow rate of 5 µl/min. Prior to injection, g5p was kept at the respective temperature of interaction. Thioredoxin was reduced immediately after immobilization and after 1015 injections of g5p with 2.5 mM DTT.
Interaction of g5p with Trx-S2Thioredoxin, immobilized on a CM5 chip through 6-amino-n-hexanoic acid, was oxidized by injecting 60 µlof 10 mg/ml 5,5'-dithio-bis(2-nitrobenzoic acid) in HBS. Association kinetics analyses were performed using freshly prepared g5p in HBS (75 µl of 40100 nM), free of DTT. Dissociation was monitored over 30 min in HBS.
Analysis of Sensorgramskd and ka were obtained by a non-linear fit of the dissociation and association parts of the sensorgram using BIA evaluation software version 2.1 supplied by the manufacturer (Biacore). The theoretical background and analysis details are described in the Biatechnology Handbook (Biacore). Bulk effect was eliminated by subtracting a control sensorgram obtained by injecting analyte (g5p) over a sensor chip surface derivatized with 6-amino-n-hexanoic acid (control), from the sensorgram, which represented interaction of thioredoxin and g5p. We have analyzed the association and dissociation phases of each experiment considering the interaction (i) homogeneous (single exponential) and (ii) heterogeneous with respect to ligand (two exponentials). Most results fitted well a model of two exponentials. KD was obtained from the ratio of kd/ka.
Thermodynamic ParametersThe Gibbs free energy change (G) was calculated from the relation
G =RTln(KA), where R is the gas constant, T is temperature in the absolute scale, and KA (1/KD) is the equilibrium association constant. R was taken as 1.9875 cal·mol1·K1. The enthalpy of complex formation or change in enthalpy (
H) was obtained from the van't Hoff equation,
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Calculation of the Activation EnergyThe effect of temperature on the reaction rate constant is given by the Arrhenius equation,
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RESULTS |
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Interaction of g5p with Trx-(SH)2 Mutantsg5p did not bind to immobilized W31H (Fig. 3A), W31A, and C35A thioredoxin (Fig. 3B). P40G Trx-(SH)2 showed very poor interaction (see Fig. 6B below), and no kinetic parameters could be obtained. Mutants D26A, W28A, W31F, W31Y, P34S, K36E, K36A, Y49F, K57M, and D26A/K57M bound with g5p but with different properties (Table II). Sensorgrams fitted well a two-exponential model apart from mutant W31F whose sensorgrams corresponded to a single-exponential model. Mutants D26A, P34S, and K36E had higher affinities to g5p when compared with wild type Trx-(SH)2.
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Interaction of g5p with Trx-S2The interaction of g5p with Trx-S2 (Fig. 4) was examined using thioredoxin immobilized through 6-amino-n-hexanoic acid. In buffers free of DTT, Trx-S2 did not interact with g5p. To demonstrate that g5p free of DTT had normal binding properties, immobilized Trx-S2 was reduced (5 mM DTT), and then its complexation with g5p free of DTT was examined. Trx-(SH)2 could bind g5p devoid of DTT. The integrity of DTT-free g5p was further confirmed by DNA polymerase assays when 100% activity was recovered after addition of DTT (data not shown).
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Thermodynamic Parameters for the Wild Type Trx-(SH)2·g5p ComplexTo monitor changes in enthalpy (H) and entropy (
S) of the Trx-(SH)2·g5p complex, binding constants were determined at 20, 25, and 30 °C (Table III). KD values increased from 1.1 to 4.3 nM while temperature increased from 20 to 30 °C, implying that the binding of g5p to thioredoxin was an exothermic reaction. The increase of KD values was mainly due to the increase in respective kd values, whereas ka values remained almost the same. Very little increase in ka values with concomitant increase in temperature indicated a low energy barrier for complex formation. No major conformation changes are therefore expected upon complexation. The plot ofln(KA) versus 1/T, gave a linear fit (Fig. 5). We assumed that the enthalpy change of the reaction (
H) would remain constant in the temperature range of the experimental, and the linearity observed in the van't Hoff plot (ln(KD) versus 1/T) indicated that this was the case. The calculated enthalpy change (
H) was20.2 kcal· M1, the Gibbs free energy change (
G) at 298 K was11.8 kcal·M1, and the entropy change for the interaction was8.4 kcal·M1. These values suggested that the complex formation of Trx-(SH)2 with g5p is driven by enthalpy and opposed by entropy. Similar observations were reported for antigen-antibody and protein-protein interactions (2127). The
G values for all mutants were less than 1 kcal·M1 with the exception of mutant W28A for which the
G value was 1 kcal·M1 (Table II).
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Activation Energy for the Wild Type Trx-(SH)2·g5p Complex FormationThe activation energy of g5p and Trx-(SH)2 was calculated from the effect of temperature on ka and kd of the complex. The plot of ln(ka) against 1/T gave a good linear fit with a root mean square deviation value of 0.976 (Fig. 6A). The calculated value of the activation energy for the forward reaction was estimated as 3.94 kcal·M1. A linear fit was obtained for the plot of ln(kd) versus 1/T with a root mean square deviation of 0.998 (Fig. 6B). The activation energy for the backward reaction was found to be 24.27 kcal·M1. The enthalpy of binding was20.23 kcal·M1, compared with 20.2 kcal·M1, which was the value obtained from the van't Hoff plot. This showed internal consistency of the results. The very fast complex formation rate between g5p and Trx-(SH)2 (ka = 4.7 x 104·M1·s1) is therefore due to a very low activation energy barrier.
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DISCUSSION |
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Reduced thioredoxin bound tightly to g5p (KD = 2.2 nM). This is because of fast association (ka = 4.7 x 104·M1·s1) and very slow dissociation (kd = 10.5 x 105·s1). In this respect, the interaction between g5p and Trx-(SH)2 is very similar to antigen-antibody interaction (30, 31). The fast association rate constant (ka), not changing with increasing temperature, indicated a low activation barrier for the complex formation and suggested no major conformational changes upon complexation. The calculated free energy change (G) at 298 K was11.8 kcal·M1. In comparison, the
G value for the lysozyme-antibody interaction was11.4 kcal·M1 and for bovine trypsin and its inhibitor was18.1 kcal·M1 (32). The plot of ln(kd) versus 1/T gave an almost straight line indicating that the enthalpy change (
H) of the reaction was insensitive to the temperature range of the experiment. The increase in KD values (Kobs) for the interaction of thioredoxin with g5p has been observed before (28).
The loss of entropy (TS) for the thioredoxin·g5p complex was 8.4 kcal·M1, whereas for most of protein-protein interactions it is about 56 kcal·M1 (32). Overall negative entropy change implies that the decrease in the translational-rotational and conformational entropies due to the thioredoxin·g5p complex formation is not fully compensated by the increase in solvent entropy. An explanation can be provided in terms of the newly ordered water molecules bound in and around the interface of the Trx-(SH)2·g5p complex leading to an overall decrease of the solvent entropy (21).
In accordance to previous studies (12), we showed that Trx-S2 was unable to induce polymerase activity to g5p, because it did not interact at all with g5p (Figs. 2 and 3). Reduced and oxidized thioredoxins are very similar in their tertiary structure. However, regions 2022, 2932, 7375, and 9394 are more mobile in reduced thioredoxin (5, 33), which can thus attain structural states not accessible to Trx-S2 (33). The crystal structure of T7 DNA polymerase shows that g5p interacts closely with the area of the active site cysteines and Arg73-Pro76 of thioredoxin. In addition, mutational studies have shown that the loops containing Gly74 (29) and Gly92 (34) are important for binding to g5p. Apparently, reduced thioredoxin can bind g5p, because it is more flexible in areas involved in the binding between the two proteins. The thioredoxin binding domain of g5p itself is also marked by increased mobility (20). From an evolutionary point of view, recruitment of only reduced thioredoxin in the T7 DNA polymerase complex could be explained in terms of the best metabolic state of E. coli supporting T7 replication. The presence of oxidized thioredoxin in the cytosol of infected cells could be an indication of oxidative stress, with the host not being ideal for phage T7 replication. Phage f1 for example does not grow in a trxB strain (35) in which the intracellular environment is known to be oxidizing (36).
The reduced state of thioredoxin, essential for its binding to g5p, may be a general prerequisite for the binding of thioredoxin to other proteins. For example only reduced mammalian thioredoxin can bind to either apoptosis signal-regulating kinase 1 (37) or thioredoxin-binding protein-2/vitamin D3 up-regulated protein 1 (38). In terms of the role of more specific amino acids in the interaction of Trx-(SH)2 with g5p, we made several observations that are given in the paragraphs that follow.
Asp26 and Lys57Asp26 is completely conserved among thioredoxins (39). The charged residue is situated inside the hydrophobic core of thioredoxin but is accessible to the solvent through a narrow channel occupied by two water molecules (7). Its carboxyl group is in close proximity to the Cys35 of Trx-S2 and Trx-(SH)2 (8) and the -amino group of Lys57 (7, 8). A D26A mutation enhanced the thermal stability of the mutant thioredoxin (33, 40, 41) but decreased its reactivity with insulin (20) and thioredoxin reductase (20, 42). Simultaneously, the pKa of Cys32 thiol was increased from 7.5 to 8.0 and the pKa of Cys35 was altered (20). It appears therefore that Asp26 stabilizes the pKa values of the active thiols of thioredoxin at appropriate values so that redox reactions can occur efficiently at physiological pH (20). Further interactions between the active site and Asp26 are demonstrated by changes in the pKa of Asp26 in reduced (pKa = 7.5) and oxidized (pKa = 9.4) thioredoxin (20). Lys57 is in proximity to Asp26 and is possibly connected with a hydrogen bond or a salt bridge to the COOH group of Asp26 (12). Lys57 could be affecting the ionization state of Asp26 and therefore the ionization state of the active site thiols (20). Oxidized and reduced thioredoxin D26A/K57M are fully folded and similar in overall structure (20).
The DNA polymerase activity was not affected by mutations at Asp26 and Lys57 (12). Kobs values of 5, 4, 5, and 6 nM have been reported for wild type, D26A, K57M, and D26A/K57M, respectively (20). The KD values obtained from SPR studies for mutants K57M and D26A/K57M were almost identical to the previous findings, whereas KD values for the wild type and mutant D26A were 2.2 and 1.7 nM (Tables I and II). These data suggest that the pKa of the active site thiols does not affect the binding of Trx-(SH)2 to g5p. The effect on binding observed for the K57M mutation was therefore not mediated through the active site thiols but most likely through some other factor. From a thermodynamic point of view, the D26A mutation (Table II) favors complex formation between g5p and thioredoxin. The conservation of an Asp at position 26 of all known thioredoxins must therefore reflect the importance of the residue for the biological function of thioredoxin.
Trp28In the crystal structure of Trx-S2, the side chain of Trp28 is packed in the hydrophobic core with its NH group forming a hydrogen bond with water (7). Trp28 is a conserved residue in prokaryotic thioredoxins (39) and is considered to increase the thermal stability of the enzyme (15). The residue is partially buried behind the disulfide bridge and is in close relation with Asp26 (43). The position of Trp28 changes significantly upon reduction (17). Trx-S2 W28A is a good substrate for thioredoxin reductase, whereas W28A Trx-(SH)2 can reduce insulin as the wild type enzyme (15). In free solution, the Kobs for thioredoxin mutant W28A and g5p is marginally higher (6 nM) than that for the wild type (4 nM). However, using SPR, Trx-(SH)2 W28A gave the highest KD (12.5 nM) of all mutants examined, mostly explainable by a 4-fold decrease in ka values. Thus, Trp28 not only increases the thermal stability of thioredoxin (15) but also enhances its binding to g5p.
Trp31Prokaryotic and eukaryotic thioredoxins and the thioredoxin domains of protein disulfide isomerase contain a conserved Trp31 exposed at the active site. Trp31 is much more mobile than Trp28 (33) and in the crystal structure of Trx-S2 is situated on the protein surface in close proximity to the Cys32 and Ile75 side chains. Its indole NH group is exposed to solvent and hydrogen-bonded to the COOH group of Asp61 (7). In the reduction of ribonucleotide reductase, the W31Y mutant was more efficient than the wild type, which was better than the mutants W31H, W31F, and W31A. A similar activity profile was observed for the reactivity of the mutants and wild type with thioredoxin reductase. In the case of the insulin assay, wild type thioredoxin was the best reductant, whereas the reactivity of the other mutants followed the previous order (Y better than H etc.) (16). These data demonstrate the requirement of a non-polar aromatic residue for position 31 of thioredoxin for the maintenance of full activity with thioredoxin reductase, insulin, or ribonucleotide reductase. In the case of g5p, Kobs values of 4, 10, 53, and 65 nM have been reported for mutants W31F, W31Y, W31A, and W31H, respectively (16). Our calculated KD values for W31F and W31Y were 3.9 and 5.2 nM, whereas replacement of Trp31 by Ala or His led to no interaction with g5p. Differences in the KD values between mutants W31Y and W31F are explainable because of higher respective ka values. The SPR data are in accordance with in vivo data. Phage T7 could not grow in a trxA strain transformed with plasmids coding for thioredoxins W31H or W31A (16). Apparently a Trp residue at position 31 of thioredoxin is vital for the interaction of the enzyme with g5p and other protein substrates.
Pro34Pro34 is exposed to the solvent, and its position changes significantly between reduced and oxidized thioredoxin (20). We expected that a mutation at position 34 would affect binding to g5p, because Pro34 is part of the hydrophobic surface area together with Ile75, Asp76, Gly92, and Ala93 (5, 6). However, P34S thioredoxin bound to g5p with a KD value of 1.6 nM, which is slightly lower than that for the wild type (2.2 nM). This is not in accordance with values described previously (28) but perhaps explains why P34S thioredoxin can support replication of phage T7 (28). P34S thioredoxin supports propagation of phage f1 as well as the wild type (35), whereas the mutant could even reduce ribonucleotide reductase better than the wild type (42). Therefore, it could be that Pro34 may not be critical for protein-protein interactions. However, the particular residue is of importance for the redox potential of thioredoxin (16).
Cys35The segment containing the active site sequence of thioredoxin (amino acids 3235) is considered to contribute to the hydrophobic surface area involved in interaction with other proteins (5, 6). The position on the SH group of Cys32 remains essentially unchanged in the structure of reduced and oxidized thioredoxin. Cys35 is known to be completely buried within the molecule in both the oxidized and reduced forms of thioredoxin (8). However, the side chain of Cys35 in Trx-(SH)2 is rotated away from the hydrophobic molecular surface implicated in binding to other proteins (6). As mentioned earlier, the thiols of the active site must be reduced for thioredoxin to bind to g5p and produce fully active T7 DNA polymerase (12). Oxidation of wild type thioredoxin or thioredoxin molecules with their thiol groups alkylated with acrylonitrile, methyl iodide, iodoacetate, and N-ethylmaleimide lead to no interaction with g5p (12). However, the active site mutant C35S, C32S/C35S, and C32A/C35S thioredoxins can bind g5p and produce the fully active T7 DNA polymerase complex (28). The Kobs for C32S/C35S was 25 nM and for C32A/C35S was 130 nM (5 nm for the wild type) (28). In view of crystallographic, genetic, and biochemical studies, Cys32 of thioredoxin is hydrogen-bonded with Thr372 of g5p in the T7 DNA polymerase complex (11). We propose that the difference in the Kobs between C32S/C35S and C32A/C35S is explainable in view of the lack of an available hydrogen in position 32 for bonding with Thr-372. SPR data showed that there is no observable interaction between g5p and mutant C35A. We propose that Cys35 of thioredoxin is also participating in a hydrogen bond with g5p, which cannot be established in a C35A mutant. Apparently a lack of a hydrogen group available for hydrogen bonding leads to no interaction of the C35A thioredoxin with g5p.
Lys36The side-chain amino group of Lys36 was originally suggested to provide a positive charge in the vicinity of Cys32 resulting in a lowering of the pKa of Cys32 (45). However, it may be that the positive charge is provided from the 2 helix dipole (42). Comparison of the x-ray crystal structures of the oxidized form of wild type thioredoxin and mutant K36E (46) shows that, although residues 2932 form a reverse turn of type I in the wild type, they flip almost 180° in the mutant. A distorted type II turn is thus formed, and the conformation of the indole ring of Trp31 is altered (46). The Glu36 of the mutant and Lys36 of the wild type occupy a similar position on the surface (46). K36E thioredoxin has a 3.5 times higher Km for thioredoxin reductase, can reduce ribonucleotide reductase as well as the wild type (42), and supports the replication of phage T7 (42). However, its binding to thioredoxin reductase was partially impaired (47). Despite the many changes in the structure of the K36E thioredoxin mentioned earlier, the KD values obtained by SPR for the interaction of K36E and K36A thioredoxin with g5p were 1.0 and 5.0 nM, respectively (2.2 nM for the wild type). The slightly lower KD value for K36E is due to an increase in the association rate constant.
P40GP40G thioredoxin is placed in the interior of 2 helix (residues 3249) where it introduces a bend of 35° (5, 6). It is most likely that replacement of Pro40 by Gly would remove the bend and result in marked structural changes. Therefore, it is not surprising that P40G thioredoxin interacted with g5p so weakly that no kinetic parameters could be obtained.
Tyr49Tyr49 is not in proximity to the active site of thioredoxin, which interacts with g5p. We expected that replacement of Tyr49 by Phe would not affect the interaction with g5p. However, the observed KD value (6.7 nM) was 3-fold higher. The elevated KD value was mostly due to reduction of the association rate constant.
The interface of intermolecular contacts is generally large (6001300 Å2) and involves 1030 side chains from each protein (4851). The free energy of the protein·protein complexes is due to strong interactions of a few specific residues or because of weaker interactions involving many residues. In the case of the human growth hormone and its receptor, the major contribution of binding energy is dependent on a few strong interactions (52, 53). In a similar manner, the binding of hen egg white lysozyme to monoclonal antibody D1.3 is determined by a small subset of residues. On the other hand, binding of D1.3 antibody to the anti-idiotype antibody E5.2 is dependent on a much larger subset of residues (44). Interactions include hydrophobic as well as polar residues. No particular relation between the hydrophobicity of the substituted specific residue and the impact on the binding constant was detected (44). In the case of thioredoxin·g5p complex, residues Trp28, Cys35 (this study), Ile75 (29), and Gly92 (34) of thioredoxin were critical for complex formation. Therefore, the interaction of the thioredoxin·g5p complex is similar to that of the growth hormone and its receptor, i.e. a few hydrophobic interactions are important for complex formation.
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FOOTNOTES |
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Present address: Immunobiology Center, Mount Sinai Medical Center, 1425 Madison Ave., New York, NY 10029.
To whom correspondence should be addressed. Tel.: 46-8-728-7686; Fax: 46-8-728-4716; E-mail: arne.holmgren{at}mbb.ki.se.
1 The abbreviations used are: g5p, gene 5 protein; DTT, dithiothreitol; ka, association rate constant; KA, association constant; kd, dissociation rate constant; KD, dissociation constant; SPR, surface plasmon resonance; Trx-S2, oxidized thioredoxin; Trx-(SH)2, reduced thioredoxin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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