Real-time Kinetics of the Interaction between the Two Subunits, Escherichia coli Thioredoxin and Gene 5 Protein of Phage T7 DNA Polymerase*

Netai C. Singha {ddagger}, Alexios Vlamis-Gardikas and Arne Holmgren §

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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T7 phage DNA polymerase is a tight 1:1 complex of the gene 5 protein (g5p) (80 kDa) of phage T7 and thioredoxin (12 kDa) from the Escherichia coli host. The holoenzyme is essential for the replication of the phage. We estimated the real-time kinetics and thermodynamics of the interaction of g5p with thioredoxin (wild type and mutants) using surface plasmon resonance. Thioredoxin was immobilized on a CM5 sensor chip through a six-carbon spacer (6-amino-n-hexanoic acid) using standard amine coupling. Reduced thioredoxin bound g5p but oxidized thioredoxin did not. The association and dissociation phases of the complex fit a two-exponential model with an apparent equilibrium dissociation constant (KD) of 2.2 nM for thioredoxin with 4.7 x 104·M1·s1 and 10.5 x 105·s1 as the corresponding association (ka) and dissociation (kd) rate constants. The strong binding of g5p to thioredoxin is therefore due to fast association and very slow dissociation, a situation similar to antigen-antibody interactions. Thioredoxin mutants P34S, D26A, K57M, D26A/K57M, W31F, W31Y, K36A, K36E, and Y49F had KD values in the range of 1 to 8 nM, whereas mutant W28A had a KD of 12.5 nM. No detectable interaction was observed for mutants P40G, W31H, W31A, and C35A. The effect of temperature on KD and the changes in enthalpy (–{Delta}H = 20.2 kcal·M–1) and entropy (T{Delta}S =–8.4 kcal·M–1) upon formation of the complex suggested that the interaction is driven by an increase in enthalpy and opposed by a decrease in entropy.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The DNA replication of bacteriophage T7 is carried out by T7 DNA polymerase, which consists of a tight 1:1 non-covalent complex of the viral gene 5 protein (g5p)1 (80 kDa) and thioredoxin (12 kDa) from the Escherichia coli host (1). g5p is a potent 3' to 5' single-strand DNA exonuclease and a weak 3' to 5' exonuclease for double-stranded DNA. Its DNA polymerase activity is low, g5p incorporating about 15 nucleotides to the primer-template before leaving it (2). Upon complex formation with thioredoxin, the affinity of g5p to primer-template increases at least 80-fold, and g5p becomes a potent polymerase (3). The 3' to 5' exonuclease activity is retained, and an additional potent 3'-5' exonuclease activity for double-stranded DNA is acquired.

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 {beta} sheets, which are surrounded by four {alpha} helices (thioredoxin fold) (4). The active site thiols of thioredoxin are located on a protruding turn between {beta}2 and {alpha}2 consisting of residues 29–37. Loop residues glycine 33, 75–76, and 91–93 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HBS buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v) surfactant P20), CM5 chip (research grade), N-hydroxysuccinimide (NHS), N-(ethyl),N'-(3-dimethylaminopropyl)carbodiimide (EDC), ethanolamine hydrochloride solution (pH 8.5), were obtained from Biacore. 6-Amino-n-hexanoic acid was from Sigma.

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 M–1·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 Thioredoxin—Surface 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 (40–60 µl of 50–100 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 (40–60 µl of 50–100 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 200–800 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 50–250 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 400–1250 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 1250–2450 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 10–15 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 40–100 nM), free of DTT. Dissociation was monitored over 30 min in HBS.

Analysis of Sensorgrams—kd 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 Parameters—The Gibbs free energy change ({Delta}G) was calculated from the relation {Delta}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 ({Delta}H) was obtained from the van't Hoff equation,

(Eq. 1)
and {Delta}H was calculated from the slope ({Delta}H/R) of a plot of–ln(KA) against 1/T. A linear plot is obtained if {Delta}H and change in entropy ({Delta}S) are not depended on temperature. {Delta}S was calculated from {Delta}G and {Delta}H using the following equation.

(Eq. 2)

Calculation of the Activation Energy—The effect of temperature on the reaction rate constant is given by the Arrhenius equation,

(Eq. 3)
where k is the reaction rate constant at temperature T, E is the activation energy, and A is the activation frequency of the reaction. Values of E were calculated from the slope (–E/R) of the linear fit of the plots of ln(ka) or ln(kd) against 1/T.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immobilization of Trx-(SH)2 and Interaction with g5p—Thioredoxin was initially immobilized directly on CM5 chips using amine coupling chemistry. Only 20% of the immobilized Trx-(SH)2 could complex g5p. Kinetic analysis of the sensorgrams using a two-exponential model gave two KD values (9.2 and 52 nM), indicating heterogeneity of the immobilized thioredoxin. The higher affinity constant was about 90% of the total SPR response. Studies employing enzymatic activity measurements have given apparent KD values (Kobs) in the area of 5 nM (20). It could be that binding of thioredoxin directly to the dextran surface hindered the interaction with g5p. To examine if this was the case, thioredoxin was immobilized on dextran through 6-amino-n-hexanoic acid, a six-carbon spacer. The resulting sensorgrams (Fig. 1) fitted well a double-exponential model (Fig. 2) and gave two sets of kinetic parameters (Table I), suggesting again a heterogeneous population of bound thioredoxin. The KD value of 2.2 nM corresponded to about 90% of the response and was chosen therefore as the "accepted" KD value. To minimize mass transport effects, only low amounts of thioredoxin (200–800 resonance units) were immobilized. The KD value obtained by SPR (2.2 nM) indicated very strong binding between Trx-(SH)2 and g5p. The result is explainable in terms of very fast association and slow dissociation. The decrease in association rate constant (ka), which was observed with increasing concentration of g5p, is reflected in the calculated KD values (Table I). It should be mentioned that g5p could not be used as an alternative immobile phase, because it binds nonspecifically to the inactivated dextran surface (data not shown).



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FIG. 1.
Interaction of wild type Trx-(SH)2 with different concentrations of g5p. Thioredoxin was immobilized on a CM5 chip through a 6-amino-n-hexanoic acid spacer. Traces 1–4 correspond to 50, 60, 80, and 100 nM g5p.

 


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FIG. 2.
Curve fitting of the association (A) and dissociation (B) sensorgrams for wild type Trx-(SH)2 and g5p. The left y-axis in A corresponds to a recorded association phase sensorgram and the resulting curve fit, shown as a superimposed continuous line. In B, the left y-axis corresponds to a sensorgram of the dissociation phase and the curve fit resulting. In both A and B, the right y-axes correspond to the residual values (dots in the diagram) for the respective curve fits. Interactions were analyzed using 50 nM g5p according to a double-exponential model.

 

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TABLE I
Kinetic parameters of the interaction of g5p with E. coli Trx-(SH)2

 

Interaction of g5p with Trx-(SH)2 Mutants—g5p 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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FIG. 3.
Sensorgrams for the interaction of g5p with Trx-(SH)2 W31A and C35A (A) and with W31H and P40G (B). Sensorgrams corresponding to plain buffer or wild type Trx-(SH)2 are shown for comparison.

 


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FIG. 6.
Arrhenius plot for the interaction of Trx-(SH)2 with g5p. Plot of forward (A) and reverse (B) reaction rate constants (ka and kd, respectively) with 1/T, where T is temperature in an absolute scale. The line represents the least-square fit.

 

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TABLE II
Kinetic parameters for the interaction of g5p with wild type Trx-(SH)2 and mutants

 

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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FIG. 4.
Interaction of g5p with Trx-S2. g5p was initially complexed with Trx-(SH)2, which was then oxidized by 5,5'-dithio-bis(2-nitrobenzoic acid). A freshly prepared solution of g5p (75 µl of 50 nM) devoid of DTT was injected, and dissociation was monitored in HBS.

 

Thermodynamic Parameters for the Wild Type Trx-(SH)2·g5p Complex—To monitor changes in enthalpy ({Delta}H) and entropy ({Delta}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 of–ln(KA) versus 1/T, gave a linear fit (Fig. 5). We assumed that the enthalpy change of the reaction ({Delta}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 ({Delta}H) was–20.2 kcal· M1, the Gibbs free energy change ({Delta}G) at 298 K was–11.8 kcal·M1, and the entropy change for the interaction was–8.4 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–{Delta}{Delta}G values for all mutants were less than 1 kcal·M1 with the exception of mutant W28A for which the–{Delta}{Delta}G value was 1 kcal·M1 (Table II).


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TABLE III
Effect of temperature on the interaction between Trx-(SH)2 and g5p

 


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FIG. 5.
van't Hoff plot for the interaction of Trx-(SH)2 with g5p. The line represents the least-square fit. {Delta}H =–20.23 kcal·M1; T{Delta}S =–8.42 kcal·M1.

 

Activation Energy for the Wild Type Trx-(SH)2·g5p Complex Formation—The 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 was–20.23 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interaction of g5p with thioredoxin has been the object of many studies (12, 20, 28, 29) in which the affinity between the two molecules has been expressed as Kobs. This is not a real KD value but stands for a set of reactions with the concentration of thioredoxin in the assay being critical for the determination of Kobs (3). The method has proved extremely useful for the comparison of the affinities of different mutants of thioredoxin to g5p (28). We used SPR in an attempt to determine "real" KD values. Preliminary SPR data showed that thioredoxin immobilized directly on the dextran surface is not accessible to g5p, and distorted binding properties were obtained. Immobilization of thioredoxin through a six-carbon spacer (6-amino-n-hexanoic acid) eliminated most steric effects and gave binding kinetics estimates (KD = 2.2 nM) in the range of those obtained for the interaction of the two proteins in solution (Kobs = 5 nM). The introduction of 6-amino-n-hexanoic acid as a spacer could perhaps facilitate more SPR studies concerning the interaction of small peptides with large molecules. For example, the method could be applicable in the interaction of peptide epitopes and their respective antibodies.

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 ({Delta}G) at 298 K was–11.8 kcal·M1. In comparison, the {Delta}G value for the lysozyme-antibody interaction was–11.4 kcal·M1 and for bovine trypsin and its inhibitor was–18.1 kcal·M1 (32). The plot of ln(kd) versus 1/T gave an almost straight line indicating that the enthalpy change ({Delta}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 (T{Delta}S) for the thioredoxin·g5p complex was 8.4 kcal·M1, whereas for most of protein-protein interactions it is about 5–6 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 20–22, 29–32, 73–75, and 93–94 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 {epsilon}-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 32–35) 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 {alpha}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 29–32 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.

P40G—P40G thioredoxin is placed in the interior of {alpha}2 helix (residues 32–49) 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 (600–1300 Å2) and involves 10–30 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.


    FOOTNOTES
 
* This work was supported by grants from the Wenner-Gren Foundation, the Swedish Research Council, and the Karolinska Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Immunobiology Center, Mount Sinai Medical Center, 1425 Madison Ave., New York, NY 10029. Back

§ 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. Back


    ACKNOWLEDGMENTS
 
We are grateful to Dr. J. Fuchs (Department of Biochemistry, Gortner laboratory, St. Paul, MN) for strain JF521 (trxA).



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