Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

Abstract

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.

Figure 1.

Stereo diagram of the 2F_o – F_c electron density map of the hGSTP1-1–GSNO complex at 1.4 Å resolution (contoured at the 1σ level). Only one conformer of the nitroso moiety is shown for clarity.

Scheme 1.

Figure 2.

Representative isothermal titration calorimetry measurements for the binding of GSNO to wt-hGSTP1-1 at 25.1°C. Titrations were performed in 20 mM sodium phosphate (pH 7.0), 5 mM NaCl, and 0.1 mM EDTA buffer. A shows the raw data for the titration of enzyme (43.66 μM) with 29 8-μL injections of GSNO (12.7 mM). A preinjection of 1 μL was performed at the beginning. (a) GSNO dilution experiment. The area under each peak was integrated and plotted in B against the molar ratio GSNO/enzyme inside the cell. The solid smooth line represents the best fit of the experimental data to a model of two equal and interacting sites (negative cooperativity).

Figure 3.

ITC data for the binding of GSNO to wt-hGSTP1-1 at 35.1°C. Titrations were performed in 20 mM sodium phosphate (pH 7.0), 5 mM NaCl, and 0.1 mM EDTA buffer. Raw data for the titration of enzyme (39.46 μM) with 29 8-μL injections of GSNO (13.95 mM). A preinjection of 1 μL was performed at the beginning. (a) GSNO dilution experiment.

Figure 4.

Temperature dependence of the global thermodynamic parameters (ΔH_g circles; −TΔS°_g, squares; and ΔG°_g, triangles) for the binding of GSNO to the C47S mutant and wild-type enzymes. The parameters are shown by hollowed and filled symbols for the C47S and the wild-type enzyme, respectively. The binding heat capacity changes were determined by linear repression analysis as the slopes of the plots of ΔH_g vs. temperature.

Figure 5.

Representative isothermal titration calorimetry measurements for the binding of GSNO to C47S-hGSTP1-1 at 29.8°C. Titrations were performed in 20 mM sodium phosphate (pH 7.0), 5 mM NaCl, and 0.1 mM EDTA buffer. A shows raw data for the titration of 35.56 μM of mutant with 29 8-μL injections of GSNO (14.1 mM). A preinjection of 1 μL was performed at the beginning. The area under each peak in A was integrated and plotted in B against the molar ratio GSNO/mutant enzyme inside the cell. The solid smooth line represents the best fit of the experimental data to a model of two equal and interacting sites (positive cooperativity).

Figure 6.

Differential scanning calorimetry transitions for the C47S mutant thermal denaturation in Buffer B, at six different scan rates: 0.2 (a), 0.5 (b), 0.83 (c), 1.16 (d), 1.33 (e), and 1.5 K/min (f). In every case, the total protein concentration is 1.12 mg/mL. DSC transitions were corrected for the instrumental and chemical baselines.

Figure 7.

Plot of ln(v/T²_m,i) vs. 1/T_m,i (i = 1, 2) (Method II). wt- and C47S-hGSTP1-1 data are shown as circles and squares, respectively. Filled and open forms represent the first (T_m,1) and the second (T_m,2) transitions, respectively (T_m,1 < T_m,2). (For details, see Materials and Methods.)

Figure 8.

Differential scanning calorimetry transitions for the thermal denaturation of wt-hGSTP1-1 (filled symbols) and C47S mutant (open symbols) in Buffer B (10 mM HEPES-NaOH at pH 7.5) at 1.5 K/min. DSC transitions were corrected for the instrumental and chemical baselines. The solid lines represent the best two-transition three-state irreversible model according to Method I. (For details, see Materials and Methods.)

Figure 9.

Excess heat capacity vs. temperature for the thermal denaturation of 1.1 mg/mL C47S-hGSTP1-1 (A) and 1.1 mg/mL wt-hGSTP1-1 (B) at 1.5 K/min. Filled and open symbols represent the experimental data in the absence and the presence of 2.1 mM GSNO, respectively. DSC transitions were corrected for the instrumental and chemical baselines. The solid lines represent the best one-transition non-two-state model. The cooperativity ratio ΔH_cal/ΔH_VH was ∼1 in all cases.

Scheme 2.