The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase

Abstract

The structure of Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TK(HSV1), measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TK(HSV1) is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TK(HSV1) in complex with two new ligands, HPT and HMTT, refined to 2.2 A are presented. Although TK(HSV1):HPT does not exhibit any significant deviations from the model of TK(HSV1):dT, the TK(HSV1):HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TK(HSV1)substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.

Fig. 1.

Conformational changes of TK_HSV1 induced by ligand binding, monitored by far-UV CD spectroscopy. The different spectra resulting from the subtraction of dialysis buffer and ligand spectra from protein spectra are shown. Spectra of TK_HSV1 (—), TK with 100 μM ATP (-.-.-), TK with 100 μM dT (. . . . .), and TK_HSV1 with 100 μM ATP and dT (-..-).

Fig. 2.

Thermal denaturation curves of ligand-free and ligated TK_HSV1 (0.4 mg/mL) in TBSE. CD signal was recorded at 223 nm in a temperature range from 20° to 70°C. (A) Influence of the natural TK_HSV1 substrates ATP and thymidine on the melting temperature. The melting curves of TK_HSV1 without substrates (♦), TK_HSV1 with 1 mM ATP (□), TK_HSV1 with 1 mM dT (X), TK_HSV1 with 1 mM ATP and 1mM dT (○) are displayed. (B) Influence of 1 mM ligand of TK_HSV1 in the presence of 1 mM ATP. The melting profiles of TK_HSV1 with ATP (♦), TK_HSV1 with HMTT:ATP (▵), TK_HSV1 with ACV:ATP (▴), TK_HSV1 with GCV:ATP (□), TK_HSV1 with HPT:ATP (·), TK_HSV1 with AZT:ATP (−), TK_HSV1 with dT:ATP (○), and TK_HSV1 with IdU:ATP (+) are shown.

Fig. 3.

CD thermal denaturation profile analysis. Fit of the CD thermal denaturation profile of TK_HSV1 (0.4 mg/mL) in TBSE containing 1 mM dT, as representative of the whole set of measured denaturation curves, to the two-state unfolding model described by Eq. 4b. (A) The open circles represent the CD signal of TK_HSV1 at 223 nm as a function of temperature. The solid line is the result of a nonlinear fit routine, fitting the unfolding CD data using Eq. 4b. The inset shows the fit results for the slope m_N and intercept Y_N of the native protein baseline, the melting temperature T_m, the enthalpy change ΔH between the unfolded and the native state (considered only as a mathematical fit parameter, not as thermodynamic value), and slope m_U and intercept Y_U of the unfolded protein baseline. (B) Residuals of the data fit shown in A representing the difference between the theoretical function and the actual data points.

Fig. 4.

Correlation between melting temperature and binding affinity by TK_HSV1. Linear relationship between the melting temperature of TK_HSV1 (0.4 mg/mL in TBSE) in complex with ATP and different TK_HSV1 substrates (each at 1 mM), and the logarithm of the K_m value of the substrates ranging over four orders of magnitude and determined by enzyme kinetics. Calibration set: IDU (K_i 0.09 μM; Balzarini et al. 1989), thymidine (K_m 0.20 μM; Pilger et al. 1999), AZT (K_m 5.2 μM; Pilger et al. 1999), GCV (K_m 47.6 μM; Kokoris et al. 1999), and ACV (K_m 200 μM; Pilger et al. 1999); test set: HPT (K_i ≅ K_m 27 μM), HMTT (K_i ≅ K_m 30.9 μM; U. Kessler, B.D. Pilger, O. Zerbe, L. Scapozza, and G. Folkers, unpubl. results). The solid line represents the linear regression calculated for the calibration set (intercept 58.47° ± 0.11°C, slope −3.52 ± 0.08 log μM, r² = 0.998, P < .001). The resulting Eq. 5 is T_melt (°C) = −3.52 * log K_m (μM) + 58.47.

Fig. 5.

Structural rearrangement monitored by fluorescence spectroscopy. Fluorescence spectra of ligand-free TK_HSV1 (solid line) and TK_HSV1 incubated with 250 μM ATP and 50 μM thymidine (gray line). Protein concentration was 0.2 mg/mL (5 μM) in TBSE containing 10 mM Tris HCl at pH 7.4, 150 mM NaCl, and 1 mM EDTA to suppress enzymatic activity. The excitation wavelength was set to 295 nm and emission fluorescence intensity was recorded at 25°C from 310 to 450 nm. Binding of ATP and dT to TK_HSV1 leads to a decrease in intensity of the fluorescence signal and a shift of the maximum from 346 to 337 nm.

Fig. 6.

Stereo view of HPT binding to TK_HSV1 compared to dT in TK_HSV1:dT complex. The conformation of HPT is well defined by the (2F_o–F_c) electron density contoured at a contour level of 1.3 σ. TK_HSV1:HPT and dT are shown in dark and light gray ball and stick model, respectively. Hydrogen bonds are depicted as dashed lines, water molecules as gray balls. dT is displayed, but the residues of TK_HSV1:dT (Champness et al. 1998) taking the same conformation as in the TK_HSV1:HPT complex were omitted for clarity. The nucleobase lies between Met128 and Tyr172 in the typical sandwich-like complex and interacts with Gln125 forming a Watson-Crick-like hydrogen- bond network as it is reported for the natural substrate dT (Wild et al. 1997; Champness et al. 1998). In addition, water-mediated hydrogen bonds between O2α and Arg176 and N1 and Tyr101 enhance the binding. The acyclic side chain is fixed similarly to the 5′-OH of dT by the interactions of the OH-group with Glu83, Arg222, and an additional water-mediated hydrogen bond with Glu225.

Fig. 7.

Superposition of TK_HSV1:HMTT and TK_HSV1:dT structures. (A) Substrate-binding site of the refined TK_HSV1:HMTT complex (subunit A) superposed with TK_HSV1:dT (Champness et al. 1998). For sake of clarity only the dT of the TK_HSV1:dT complex is displayed. The conformation of HMTT is well defined by the (2F_o–F_c) electron, shown at a contour level of 1.3σ in blue. The carbon atoms of TK_HSV1:HPT and dT are shown in dark and light gray, respectively, whereas the other atoms are color-coded (N: blue, O: red, S: yellow). Hydrogen bonds are shown as dashed lines, water molecules as green balls. (B) View into the active site showing a displacement induced by HMTT binding. TK_HSV1:HMTT and TK_HSV1:dT are shown in yellow and gray, respectively. All residues of TK_HSV1:dT taking the same conformation as in TK_HSV1:HMTT complex were omitted for clarity. Hydrogen bonds and water are displayed as in A. The thymine part of HMTT is positioned like the base of the natural substrate dT, whereas the sugar-mimicking moiety substituted at position 6 of the thymine ring is inserted much deeper into the active site. The 1.4-Å shift of the sulfate ion toward the substrate and the direct hydrogen bond between the hydroxyl group of HMTT mimicking the O5′ of dT is shown. The rearrangement occurring for the amino acid residues 60–75 of the P-loop and the displacement of the following helix α1 is depicted.