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J. Biol. Chem., Vol. 275, Issue 21, 16139-16145, May 26, 2000

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Compulsory Order of Substrate Binding to Herpes Simplex Virus Type 1 Thymidine Kinase

A CALORIMETRIC STUDY^*

Remo Perozzo, Ilian Jelesarov^§, Hans Rudolf Bosshard^§, Gerd Folkers, and Leonardo Scapozza^¶

From the  Department of Applied Biosciences, Swiss Federal Institute of Technology, Winterthurerstr. 190, CH-8057 Zurich, Switzerland and the ^§ Department of Biochemistry, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland

Received for publication, January 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK·dT complex. At pH 7.5 and 25 °C, the binding constants are 1.9 × 10⁵ M¹ for dT and 3.9 × 10⁶ M¹ for ATP binding to the binary HSV1 TK·dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (Cp) obtained from H in the range of 10-25 °C is 360 cal K¹ mol¹ for dT binding and 140 cal K¹ mol¹ for ATP binding. These large Cp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of Cp and TS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK·dT·ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Molecular recognition phenomena are at the heart of biological reactions. Key to the understanding of molecular recognition is a comprehensive analysis of the thermodynamics of binding and a meaningful correlation of thermodynamics with structure. A close insight into the thermodynamics of a binding process provides guide marks for structure-based molecular design strategies. The forces that govern a binding reaction are the free energy change (G), the enthalpy change (H), the entropy change (S), and the heat capacity change (Cp). Cp is an approximate measure of the surface area buried in an association reaction and can be used to predict conformational rearrangements in associating protein molecules. An example of high medicinal interest where such information is essential is thymidine kinase from herpes simplex virus type 1 (HSV1 TK).¹ The structure of this enzyme is known at high resolution in complex with a series of ligands, including various substrates (natural and non-natural) and inhibitors (1-5).

Thymidine kinases (EC 2.7.1.21) catalyze the phosphorylation of thymidine (dT) to dTMP in the presence of magnesium ions by transferring the -phosphate group of ATP to the 5'-OH group of dT. Herpesviruses encode their own thymidine kinases, which differ considerably from the enzyme of the human cellular host (human cellular thymidine kinase). While the human enzyme is highly specific, HSV1 TK is a multifunctional enzyme of broad substrate specificity. It shows deoxycytidine kinase and thymidylate kinase activity (6) and phosphorylates a broad spectrum of pyrimidine as well as purine analogs (7-12). Moreover, HSV1 TK displays low stereochemical specificity. The enzyme accepts modified ribose moieties, acyclic side chains, and the L-stereoisomer of the deoxyribose of dT (13). The preferred phosphate donor is ATP, yet HSV1 TK also shows high affinity for cytidine triphosphate, uridine triphosphate, and guanosine triphosphate and their deoxy analogs.

Therapeutic applications involving HSV1 TK make use of the broad substrate diversity of the viral enzyme in the background of strict substrate selectivity of the host cell enzyme. Therefore, a detailed thermodynamic analysis of substrate binding to the viral kinase is a prerequisite for the successful design of new therapeutically useful compounds.

HSV1 TK is a homodimer with 376 residues per subunit (Fig. 1). The constituent subunits display the general / folding pattern. A central five-stranded parallel -sheet is flanked on either side by helices. HSV1 TK is a member of the family of NMP kinases and contains the classical mononucleotide (NMP) binding fold (14). In this enzyme family, three-dimensional structures are known for adenylate kinase (15, 16), guanylate kinase (17), uridylate kinase (18), bacteriophage T4 deoxynucleotide kinase (19), and thymidylate kinase (20). The central five--strand domain is referred to as the CORE domain. Other domains are the LID domain and the NMP_bind domain (21). Main chain superposition of HSV1 TK and adenylate kinase reveals substantial similarity in the CORE domain. Major differences exist in the NMP_bind domain, where HSV1 TK has extensive insertions. The LID domain of HSV1 TK consists of only eight residues, reminiscent of other small variants of NMP kinases. Further differences from adenylate kinase arise from the following unique structural features of HSV1 TK: a 45-residue-long amino-terminal segment, which is not resolved in the crystal structure and is not necessary for catalytic activity (22) but plays a role in migration within the cell (23); a partially mobile segment of 72 residues between residues 250 and 322; and an additional 29 C-terminal residues (2). Interestingly, many of these differences appear to be located close to the dimer interface.

View larger version (80K): [in this window] [in a new window]   Fig. 1.   Ribbon diagram of the symmetric HSV1 TK dimer with bound ADP and dTMP (Protein Data Bank entry 1VTK; Ref. 2). The domains are defined as for other NMP kinases (21). The CORE domain is depicted in blue, the NMPbind domain in red, and the LID domain in yellow. The additional residues (250-322) are shown in green. Substrate and cofactor are depicted as cyan ball and stick models. The image was generated with the program MOLSCRIPT (53).

Adenylate kinases undergo large conformational changes upon substrate binding as shown by crystallography (24). The substrate-free enzyme has a more open conformation, and substrate binding leads to a closed conformation. The NMP_bind domain and the LID domain rearrange upon binding of AMP and ATP, whereas the conformation of the CORE domain remains unchanged (21, 24). The three-dimensional structures of HSV1 TK known to date have been solved for ternary complexes with natural and non-natural substrates, inhibitors, cofactors, or sulfate ions mimicking the -phosphate of ATP (1-5) and correspond to the closed conformation of NMP kinases. The structure of the free apoenzyme is not known. Since structural similarities in the CORE domain and substrate binding pockets are substantial in the NMP kinase family, one may assume that substrate-free HSV1 TK also exists in an open conformation and that conformational changes take place when HSV1 TK is converted to the closed conformation during substrate binding.

Here we present a comprehensive thermodynamic analysis of nucleoside (dT) and cofactor (ATP) binding to HSV1 TK. Substrate binding, which was followed by ITC, is shown to be strictly sequential, with dT binding first and ATP second. Combining the thermodynamic parameters with the three-dimensional structure of the ternary HSV1 TK·dT·ATP complex demonstrates that the sequential binding pathway is accompanied by significant structural rearrangements of the enzyme.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Materials-- All chemicals were of analytical grade and were used without further purification. Thymidine, ATP, glutathione, buffer reagents, and glutathione-agarose (SH-coupled via 12 C-spacer) were from Sigma; dithiothreitol and EDTA were from Fluka.

Expression and Purification-- The bacterial expression vector pGEX2T-TK was constructed as described previously (25, 26). HSV1 TK was expressed as glutathione S-transferase fusion protein (GST-TK) in Escherichia coli strain BL21 and purified by glutathione affinity chromatography (25). After isolation of the fusion protein from the crude extract by glutathione-affinity chromatography, the protein was directly (on column) exchanged into the experimental buffer by thoroughly rinsing the column with an excess of buffer. The protein was eluted by the addition of 5 mM glutathione and used for titration experiments. The total protein concentration was determined using a dye-binding assay (27). The concentration of active enzyme present in the protein samples used for ITC was determined by ATP affinity chromatography in the presence of dT (25). Inactive enzyme does not bind to this column. The amount of inactive enzyme was 20-30% of the total protein concentration and was corrected for in the calculation of the binding constants (K_B) and enthalpy of binding (H) from ITC raw data.

Isothermal Titration Calorimetry-- ITC experiments were carried out using an OMEGA titration microcalorimeter (Microcal Inc., Northampton, MA) equipped with a nanovolt preamplifier to reduce electrical noise (28). The reference cell was filled with water containing 0.01% sodium azide, and the calorimeter was calibrated using standard electrical pulses as recommended by the manufacturer. All solutions were degassed for 10 min with gentle stirring under vacuum. Solutions of the fusion protein were filled in the sample cell (1.34-ml volume) and titrated with dT or ATP. Substrate solutions were prepared in the buffer from the final step of protein purification. The substrate concentration in the injection syringe was usually 25 times higher than the concentration of protein binding sites. A typical experiment consisted of a first control injection of 1 µl followed by 19 injections, each of 4 µl and 15 s duration, with a 4-min interval in between.

ITC measurements were routinely performed in 50 mM Tris, pH 7.5, 4 mM EDTA (to suppress enzymatic activity), 5 mM glutathione, 1 mM dithiothreitol. Heat contributions due to coupled protonation events upon binding were evaluated by calorimetric experiments in various buffers of different ionization enthalpies under otherwise identical conditions. The buffers and their ionization enthalpies (in kcal mol¹ at 25 °C) were as follows: PIPES (2.7), MOPS (4.9), TES (7.7), and Tris (11.34) (29). The pH of the buffer was adjusted at the experimental temperature. Buffer concentrations were 50 mM, and the ionic strength was similar for all buffers. In control experiments, the ligand was injected into buffer. The observed heat effects were concentration-independent and were identical to the heat signals detected after complete saturation of the protein. Therefore, the nonspecific background was usually estimated by averaging the small heats at the end of the titration. Raw data were collected, corrected for ligand heats of dilution, and integrated using the Microcal Origin software supplied with the instrument. Since protein concentration was expressed on a subunit basis, a single-site binding model was fit to the data by a nonlinear regression analysis to yield binding constants (K_B), enthalpies of binding (H), and stoichiometry of binding.

HPLC Assay-- High performance liquid chromatography was used for concentration determination of thymidine and ATP in the final ligand solutions and to monitor potential phosphorylation products during calorimetric experiments using a modified protocol of a previously published method (30). Nucleotides were determined by reverse-phase ion-paired chromatography using a C₁₈ column (LiChrospher 100 RP-18, 5 µm, 250 × 4 mm; Merck) in 0.2 M NaH₂PO₄, 25 mM tetrabutylammonium, 3% (v/v) methanol at 1.0 ml/min and detection at 254 nm. Ligand concentrations were calculated by means of calibration curves from standard solutions showing linearity in the range of <0.04 to at least 2 mM. The detection limit for dT, dTMP, and ATP was <20 nmol (31).

Calculation of Solvent-accessible Surface Area-- The program NACCESS (32), an implementation of the Lee and Richards solvent accessibility algorithm (33), was used with a probe radius of 1.4 Å and a slice width of 0.25 Å. ASA_ap and ASA_pol were calculated using the coordinates of the ternary complex TK·dT·ATP (1) and removing either dT or ATP or both.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Experimental Setup-- The GST fusion protein of HSV1 TK was used in this study to facilitate protein purification and to improve stability during storage. To rule out artifacts caused by the presence of GST in the construct, control experiments were performed with the HSV1 TK obtained after on-column cleavage of the affinity tag. Thermodynamic parameters determined by ITC were identical within error for cleaved HSV1 TK and for the GST fusion protein (data not shown). The influence of glutathione in concentrations up to 5 mM on HSV1 TK kinetics was investigated as previously described (25, 34). Glutathione in concentrations up to 5 mM does not change K_m (data not shown). Since Mg²⁺ is strictly required for the phosphorylation reaction catalyzed by HSV1 TK, all measurements were performed in the presence of 4 mM EDTA to suppress any enzymatic activity in ITC experiments. Since Mg²⁺ could influence the equilibrium binding parameters by either direct participation in the bonding network of the binding pocket or by indirect structural effects, control titrations with the noncleavable ATP derivative ,-methyleneadenosine 5'-triphosphate were performed in the presence and absence of Mg²⁺. No differences were seen (data not shown).

Determination of the Thermodynamic Parameters of dT and ATP Binding by ITC-- In the ternary complex TK·dT·ATP, the substrate dT and the cofactor ATP are located in separate and well defined binding pockets of the enzyme. Formation of the ternary enzyme-substrate complex may either proceed through an obligatory sequential pathway or by a random mechanism. Two sequential pathways are possible: TK  TK·dT  TK·dT·ATP (reactions i and ii shown in Fig. 2) or TK  TK·ATP  TK·dT·ATP (reactions iii and iv in Fig. 2). In a random mechanism, binding of one substrate is not a prerequisite for binding of the other, and all four reactions of Fig. 2 can take place.

View larger version (8K): [in this window] [in a new window]   Fig. 2.   Formation of the ternary enzyme-substrate complex TK·dT·ATP. The two ordered sequential pathways are i plus ii and iii plus iv, respectively. In a random binding mechanism, all four reactions take place.

To distinguish between ordered and random binding, HSV1 TK was titrated with dT and with ATP, respectively. Fig. 3 shows representative titrations of the substrate-free enzyme with dT (A and B) and with ATP (C and D). The titration with dT was characterized by a significant exothermic heat effect. In Fig. 3B, the integrated heat of each injection (filled squares) is plotted against the molar ratio of dT to enzyme binding sites, and the solid line is a nonlinear least squares fit for a single-site binding model. The mean values of the thermodynamic parameters at 25 °C and pH 7.5 for the titration of the substrate-free enzyme with dT, resulting from three independent measurements, are K_B = 1.9 ×10⁵ M¹ and H_bind = 19.1 kcal mol¹ (Table I).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Representative isothermal titration calorimetry measurements. A and B, titration of HSV1 TK with dT, corresponding to reaction i of Fig. 2. C and D, titration of TK with ATP, corresponding to reaction iii in Fig. 2. E and F, titration of TK with ATP in the presence of excess dT, corresponding to reaction ii of Fig. 2. G and H, titration of HSV1 TK with a 1:1 mixture of dT and ATP. Raw data are shown as differential power signals in A, C, E, and G. Binding isotherms obtained by integration and normalization of the raw data and by correction for the heat of ligand dilution are shown in B, D, F, and H. The solid lines represent nonlinear best fits for a single-site binding model. Titrations were performed in 50 mM Tris/HCl, pH 7.5, 25 °C. A and B, 60.6 µM enzyme was titrated with 2.2 mM dT. C and D, 51 µM enzyme was titrated with 1.3 mM ATP. E and F, 37.3 µM enzyme in the presence of 1 mM dT was titrated with 1.4 mM ATP. Under these conditions, >99% of the enzyme was present as TK·dT complex. G and H, 51.5 µM enzyme was titrated with a 1:1 mixture of 1.6 mM dT and ATP. For the purpose of clarity, only one representative result of triplicate measurements is shown. Values on the y axis (kcal/mol of injectant) are raw data and are not yet corrected for protonation effect (Equation 1).

View this table: [in this window] [in a new window]   Table I Thermodynamic parameters for the binding of dT and ATP to HSV1 TK at pH 7.5  Values of H are corrected for protonation effect by Equation 1. Values of G, H, and TS are in kcal mol1. Cp is in kcal K1 mol1. Values are the mean of triplicates. G was calculated from G = RT × ln KB, where KB is the binding constant determined by ITC. Uncertainty of G is within ±0.35 kcal mol1 of the mean. Errors of H are about ±5% and mainly reflect the error in ligand concentration. Maximal possible errors of TS are 1.5 kcal mol1. Errors of Cp were estimated by reduction of the data set by one data point at a time and were, on average, ±0.02 kcal K1 mol1, i.e. within 5-15% of the reported mean.

Titration with ATP showed a very different behavior (C and D). Only very small heat signals corresponding to nonspecific heat effects were detected. Titrations were repeated at 15 °C and at varying concentration ratios of ATP to enzyme. No heat effect typical of a binding reaction was observed (data not shown). This indicates that empty HSV1 TK does not bind ATP. Binding driven by entropy without heat change could be excluded (see below). In agreement with the inability of the apoenzyme to bind ATP is the fact that thymidine-depleted HSV1 TK did not bind to ATP affinity columns, even at very high protein concentrations (25).

To confirm an ordered binding mechanism in which TK·dT precedes the formation of TK·dT·ATP, the experiment shown in Fig. 3, E and F, was performed. The preformed TK·dT binary complex (1 mM dT) was titrated with ATP (reaction ii in Fig. 2). The reaction was again exothermic and yielded values of K_B = 3.9 × 10⁶ M¹ and H_bind = 13.8 kcal mol¹ (Table I; corrected for protonation effect; see below).

In the cell, HSV1 TK is exposed to both substrates at the same time, which in the scheme of Fig. 2 corresponds to a move on the diagonal from TK to TK·dT·ATP. If the binding reaction is indeed sequential as expected from the above experiments, titration of TK with a 1:1 mixture of dT and ATP should yield a heat change corresponding to the sum of the heat changes for reactions i and ii of Fig. 2. A representative titration is shown in Fig. 3, G and H. The titration isotherm yielded H_bind = 33.1 kcal mol¹ (Table I, corrected for heat of protonation), identical within error to the sum of the enthalpy changes of reactions i and ii.

The ITC experiment provides the binding constant K_B for a single-site reaction, and G values of reactions i and ii were calculated from G = RT × ln K_B. S was obtained from G = H  TS. Reactions i and ii were driven by favorable negative changes in binding enthalpy and strongly opposed by unfavorable entropic contributions. Although the reaction with the 1:1 mixture of dT and ATP was more complex, it could still be treated as a single-site reaction if one considered dT plus ATP as one ligand. In this case, K_B obtained from ITC equaled (K_i × K_ii)^1/2 where K_i and K_ii were the binding constants for reactions i and ii, respectively. Hence, the apparent binding constant for the coupled reactions i and ii was K_B², and G equaled RT × ln K_B².

Change of Protonation State-- Substrate binding may cause the enzyme to take up or release protons, for example through pK_a changes of side chains accompanying the binding reaction. This will contribute to the overall heat change, H_obs, measured in the ITC experiment. If ionizable groups undergo pK_a changes on complex formation, protons will be exchanged with the buffer. The heat of protonation/deprotonation depends on the ionization enthalpy of the buffer, H_ion, according to the equation,

(Eq. 1)

where n_H+ designates the number of protons that are released (n_H+ >0) or taken up (n_H+ < 0) by the buffer (35). To study such protonation effects, titration experiments were repeated in various buffers of different H_ion. The intrinsic enthalpy of binding, H_bind, was obtained from the intercept (H_ion = 0) of a plot according to Equation 1. The results are shown in Fig. 4. Protonation/deprotonation was negligible in the case of dT binding to the free enzyme (Fig. 4, line B). Hence, H_obs = H_bind for reaction i. An uptake of 0.31 protons was observed with ATP binding to the TK·dT complex in reaction ii (Fig. 4, line A). Titration with the 1:1 mixture of dT and ATP leads to the uptake of 0.35 protons (Fig. 4, line C). It follows that proton uptake occurred with ATP binding but not with dT binding. Heat changes from ITC experiments were corrected accordingly (Table I).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Protonation effect. Experimentally observed enthalpy change, Hobs, is plotted versus the ionization enthalpy of the buffer, Hion, at 25 °C to evaluate the protonation effect. ITC experiments were performed at pH 7.5 in PIPES, MOPS, TES, and Tris buffers. A, titration of enzyme with ATP in the presence of excess dT (reaction ii in Fig. 2). B, titration of enzyme with dT (reaction i in Fig. 2). C, titration of enzyme with a 1:1 mixture of dT and ATP. Continuous lines are linear least-squares fits according to Equation 1.

Phosphate buffer behaved anomalously and influenced the thermodynamic parameters significantly. Therefore, data collected in phosphate buffer were not included in the analysis because of obvious differences in the interaction mechanism.

Temperature Dependence of Thermodynamic Parameters-- ITC measurements were performed at 10, 15, 20, and 25 °C. The results are presented in Fig. 5 and are summarized in Table I. H and TS depended strongly on temperature, while G was almost insensitive to temperature due to enthalpy-entropy compensation. Values of Cp were calculated from the slopes of the regression lines of H_bind versus temperature (Fig. 5). Binding of dT to the free enzyme (reaction i) was characterized by Cp = 360 cal K¹ mol¹. Cp = 140 cal K¹mol¹ was measured for ATP binding to the TK·dT complex (reaction ii), and Cp = 510 cal K¹ mol¹ for the titration of the enzyme with a 1:1 mixture of dT and ATP (Table I). The latter value was very close to the sum of the Cp values of reactions i and ii, in agreement with a thermodynamic cycle described by the three reactions.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Temperature dependence of H, TS, and G. ITC experiments were performed in Tris/HCl, pH 7.5. A, binding of dT to the free enzyme (reaction i). B, binding of ATP to the enzyme presaturated with dT (reaction ii). C, binding of a 1:1 mixture of dT and ATP to the free enzyme. Cp was obtained from the slope of a linear least-squares fit to H data. H values are displayed as triangles, while G and TS values are shown as squares and diamonds, respectively. Reported data are corrected for protonation effect by Equation 1.

Correlation between Cp and Surface Area Buried on Substrate Binding-- In protein folding, the changes in enthalpy, entropy, and heat capacity can be accounted for in terms of changes in solvent-accessible polar and apolar surface area (36-43). Since changes in the atomic interactions are similar in protein folding and in binding reactions involving proteins (44), thermodynamic parameters of protein-protein, peptide-protein, and small ligand-protein interactions can be related to changes in solvent-accessible surface in the same way (45, 46). Cp scales with the amount and type of surface buried in the complex according to the equation,

(Eq. 2)

where ASA represents the apolar (ap) and polar (pol) surface buried in the complex, and C represents the elementary contributions per Ų of apolar and polar surface to the heat capacity and enthalpy changes (45).

Since there are no structural data available for HSV1 TK in the ligand-free state, to estimate ASA, one may remove dT and ATP from the crystal structure of the ternary complex to obtain ASA. The buried surface area calculated in this way corresponds to a rigid body binding model in which no conformational changes take place when dT and ATP bind. As seen in Table II, ASA_tot calculated for the rigid body model was on the order of 600 to 800 Ų for both dT binding and ATP binding. However, values of Cp calculated by Equation 2 using the rigid body values of ASA were much smaller than Cp determined by experiment (Cp_calc and Cp_exp in Table II).

View this table: [in this window] [in a new window]   Table II Analysis of the changes of solvent-accessible surface area Calculated changes of solvent-accessible surface area (ASA) caused by substrate binding to HSV1 TK under the assumption of a rigid body binding model are shown. Based on this assumption, ASA have been calculated by removing dT, ATP, or both from the crystal structure of the TK·dT·ATP complex (1). ASA is in Å2 and Cp in cal K1 mol1. Cpcalc values have been calculated from Equation 2 with ASA calculated from the crystal structure of TK·dT·ATP (rigid body assumption). Cpexp values are from Table I.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Ordered Binding of Thymidine and ATP to HSV1 TK-- The calorimetric titrations presented here provide a comprehensive description of the energetics of substrate binding to HSV1 TK. Substrate binding is strictly ordered. ATP could bind only after the TK·dT complex had been formed. No ATP binding to the apoenzyme was observed. One might argue that ATP binding was entropy-driven and that H for ATP binding was so small that it escaped detection by ITC. This possibility could be ruled out because the entire set of thermodynamic parameters satisfied the cycle shown in Fig. 6. Fig. 6 summarizes the energetics of binding of dT and ATP at 25 °C and pH 7.5. Summation of the parameters for reactions i and ii equaled almost exactly the parameters determined independently for the single reaction of the apoenzyme with dT and ATP together. The free energy change for dT binding was smaller than for ATP binding, 7.2 versus 9.0 kcal mol¹, but the enthalpy, entropy, and heat capacity changes were significantly larger for the initial dT binding. Comparing absolute values, one notes that H was 1.4 times larger, and TS and Cp were 2.5 times larger for dT binding than for the subsequent ATP binding. This means that dT binding, which induced the formation of a tight ATP binding site, was driven by a large enthalpy change but at a high cost in entropy.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Thermodynamic cycle for sequential binding of dT and ATP to HSV1 TK. The parameters at 25 °C and pH 7.5 are indicated (G, H, and TS in kcal mol1; data from Table I).

Substrate-induced Conformational Changes Deduced from Large Cp Values-- In the relatively narrow temperature range studied, H became more favorable and TS more unfavorable with increasing temperature. As a result, G remained remarkably insensitive to temperature through entropy/enthalpy compensation. This is a ubiquitous phenomenon seen in many association reactions and is thought to be directly related to the role of solvent water molecules in the association process (47). According to the laws of thermodynamics, the temperature dependence of H and S results from substantial changes in heat capacity. In almost all association processes with proteins, Cp has a negative sign if the free components are the reference state (45). In the present case, Cp of the overall reaction was 510 cal K¹ mol¹. Binding of dT contributed 70%, and ATP binding contributed 30% to this large negative heat capacity change. Recently, it has been shown that the enthalpy slope observed in ITC experiments may include contributions arising from temperature-induced changes in the heat capacities of all participants of the association reaction over the considered temperature range (48). The quantitative information that is needed to correct the experimental Cp for such effects could be obtained by DSC, given that the individual components undergo a reversible thermal unfolding. Unfortunately, the thermal unfolding of HSV1 TK was fully irreversible, with an apparent midpoint at 43 °C. However, the far and near CD spectra remained unchanged, and the enzyme retained full enzymatic activity over the relatively narrow temperature interval of this study. We believe, therefore, that the measured H and Cp values genuinely reflect the formation of the specific ligand-protein complex, including the structural rearrangements of the enzyme to accommodate the ligands and do not contain significant contributions from preexisting temperature-dependent conformational equilibria.

Since, in general, Cp correlates well with the amount of surface area buried at the complex interface (49-52), we have tried to calculate Cp from the surface area of HSV1 TK buried by dT and ATP, respectively, and by both substrates together. The calculation (Equation 2) is based on a large body of structural and thermodynamic data for protein folding, protein-protein association, and protein-ligand binding.

Since the three-dimensional structure of substrate-free HSV1 TK is not known, we could not relate the calculated surface changes with actual structural data. However, from the crystal structure of the TK·dT·ATP complex, one can calculate the buried area for the case of a rigid body binding model in which the dT and ATP binding sites preexist and no conformational changes take place. Obviously, this assumption was unjustified in view of the ordered sequential binding mechanism. Indeed, the heat capacity changes calculated from ASA for the rigid body binding model were accordingly much smaller than the experimentally determined ones (Table II).

One has to remember that surface burial calculated according to Equation 2 takes into account mainly hydration effects, which are not the only contributions to Cp. Changes in molecular vibration modi are influenced by ligand binding in a deep hydrophobic binding pocket, as seen in the crystal structure of the TK·dT·ATP complex, in which the binding sites are close to the interface between the tightly associated monomers of the functional dimer. Hence, vibrational effects may have contributed to the large negative Cp values in addition to hydration effects, but they were not considered in the semiempirical analysis. Nevertheless, the significant discrepancy from a rigid binding model is an indisputable sign of significant conformational rearrangements accompanying substrate binding to HSV1 TK. Indeed, in the crystal structure of the ternary enzyme-substrate complex, dT is deeply buried and completely caved due to closing of the LID domain (Fig. 1). The ATP binding site is more surface-exposed, with ATP located like a plug just in front of dT (1). Thus, a large contribution by conformational rearrangements would seem to be justified. Reorganization of the dimer interface concomitant to substrate binding would also seem to be feasible. The dT binding site in the ternary complex of HSV1 TK is localized in a large pocket of the NMP_bind domain that is lined up by helices ₃, ₄, and ₅. These elements also belong to the dimer interface, and one might assume that the association of dT and the subsequent formation of the ATP binding site could be communicated to the dimer interface. In addition, parts of the CORE domain and of the mobile segment between residues 250 and 322 contribute to the NMP_bind domain. While these considerations remain speculative before the structure of substrate-free HSV1 TK is known, recent crystallographic studies of adenylate kinases, to which the viral thymidine kinase is homologous, have indeed demonstrated major changes induced by substrate binding in the NMP_bind domains and the LID domains as well as small changes in the CORE domains (21, 24).

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

This is the first report providing a comprehensive thermodynamic description of substrate and cofactor binding to HSV1 TK, a representative of the large family of nucleotide and nucleoside kinases. The results obtained by titration microcalorimetry reveal an extreme case of positive heterotropic interaction. Formation of a binary complex of thymidine with HSV1 TK is a stringent prerequisite for ATP binding. Since the ATP binding site is in fact generated by thymidine binding, one would expect the enzyme to undergo considerable conformational rearrangements. This has been supported by the analysis of the observed heat capacity changes, which were large and negative and indicated burial of molecular surface to an extent much larger than expected if the substrate binding sites preexisted on the apoenzyme and no rearrangement would occur (rigid body binding model). The findings support the view that substrate binding to HSV1 TK leads to a conformational closing of the substrate binding sites to bring thymidine and ATP into an orientation appropriate for catalysis. The details of the predicted rearrangements must await a firm structural foundation. Work is in progress in this laboratory to solve the crystal structure of the substrate-free apo form of HSV1 TK.

	FOOTNOTES

^* This work was supported in part by grant from the Swiss National Science Foundation and the Canton of Zurich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 41-1-635-6036; Fax: 41-1-635-6884; E-mail: scapozza@pharma.ethz.ch.

Published, JBC Papers in Press, March 13, 2000, DOI 10.1074/jbc.M000509200

	ABBREVIATIONS

The abbreviations used are: HSV1, herpes simplex virus type 1; TK, thymidine kinase; dT, thymidine; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; ITC, isothermal titration calorimetry; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; NMP, nucleoside monophosphate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

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