Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

Abstract

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD(+) synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, K(a), equal to 5.6 x 10(6)/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer <==> folded monomer <==> unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K(a) down to approximately 10(6)/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.

Figure 1.

Two consecutive DSC scans of the same NAD⁺ synthetase sample. (a) First scan; (b) second scan. Total protein concentration: 1 mg/mL; Buffer: 60 mM EPPS at pH 8.5, 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂, without DMSO. Scan rate: 1.1°C/min.

Figure 2.

DSC curve fitting of NAD⁺ synthetase thermal unfolding. (A,B) DSC scan of NAD synthetase (0.8 mg/mL) in 60 mM EPPS (pH 8.5), 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂. Scan rate: 1.5°C/min. (A) The DSC data (solid line) is shown in comparison to the fit to a two-state model (dashed line). (Inset) Residual errors in excess heat capacity for the two-state fit of the DSC data. (B) The DSC data (solid line) is shown in comparison to the fit to a two-state dissociative model, using the entire DSC data (dashed line). Circles represent the fit of the first half of the DSC data (up to T_1/2). (Inset) Residual errors in excess heat capacity for the two-state with dissociation fit of the DSC data. (C,D) DSC scan of NAD⁺ synthetase (0.9 mg/mL) in 60 mM EPPS (pH 8.5), 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂, 2.5% (v/v) DMSO. Scan rate: 1.5°C/min. (C) The DSC data (solid line) is shown in comparison to the fit to a two-state dissociative model (dashed line). (Inset) Residual errors in excess heat capacity for the two-state dissociative fit of the DSC data. (D) The DSC data (solid line) is shown in comparison to the fit to a sequential three-state model using the entire DSC data (dashed line). Circles represent the fit of the first half of the DSC data. (Inset) Residual errors in excess heat capacity for the sequential three-state fit of the DSC data.

Figure 3.

Scan-rate dependence of the apparent van’t Hoff enthalpy (ΔH_v,app). Plots of ΔH_v,app vs. the reciprocal of the scan rate (1/v) for NAD⁺ synthetase unfolding without (open circles) and with (solid circles) DMSO. (Open circles) NAD⁺ synthetase (~1 mg/mL) in 60 mM EPPS (pH 8.5) with 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂. Data fitting used the two-state dissociative model. The extrapolated ΔH_v value at infinite scan rate is 953 (±17) kJ/mole. The rightmost point was excluded from the linear regression. (Solid circles) NAD⁺ synthetase in 60 mM EPPS (pH 8.5) with 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂, 2.5% DMSO. Data fitting used the sequential three-state fit model. The extrapolated ΔH_v for unfolding (Equilibrium 2; see Materials and Methods for details) at infinite scan rate was 487(±10) kJ/mole. The calculated total ΔH_v for both equilibria is 1151 (±17) kJ/mole (see Table 2).

Figure 4.

Dependence of T_1/2 (in Kelvin) on the total protein concentration, for NAD⁺ synthetase unfolding without DMSO. T_1/2 values for 0.265, 0.388, 0.53, and 0.88 mg/mL Pt were included. Buffering condition: 60 mM EPPS at pH8.5, 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂. The straight line has a slope of −1.03 (±0.1) × 10⁵ K, which corresponds to a ΔH_v of 858 (±84) kcal/mole; see text for details.

Figure 5.

DMSO effect on the dimerization equilibrium of NAD⁺ synthetase measured by sedimentation equilibrium at 20°C and 12500 rpm. (Bottom curve) 0.75 mg/mL protein in 60 mM EPPS (pH 8.5) with 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂, without DMSO. The curve defined by “+” symbols is a mathematical fit using a single ideal species model. (Inset A) Residual plot of the fit. The calculated molecular weight of the species is 61.1 ± 1.4 kD (average of 6 runs), which corresponds to the molecular weight of the NAD⁺ Synthetase homodimer. (Top curve) 0.75 mg/mL protein in 60 mM EPPS (pH 8.5) with 20 mM KCl, 19 mM NH₄Cl, 10 mM MgCl₂, 2.5% (v/v) DMSO. The curve defined by open circle symbols is a mathematical global fit to all 6 runs performed at 20°C, using the self-association model. (Inset B) Residual plot of the fit. The calculated association constant (K_a) for dimerization is (5.6 ± 0.02) × 10⁶/M.

Figure 6.

Dissociation constant of the NAD⁺ synthetase dimer (K_d) in the presence of 2.5% (v/v) DMSO as a function of the reciprocal of temperature. The smooth curve is a fit for the data, using a simple van’t Hoff analysis (equation 14; see Materials and Methods for details). The van’t Hoff enthalpy (ΔH_v1) from the fit is 113.3 (±10) kJ/mole at T = 39.0°C and the change in heat capacity (ΔCp₁) is 4.67(±0.8) kJ/(mole-K). Each data point represents the K_d calculated from a global fit of six different experimental runs at temperatures 15°, 20°, 25°, 35°, and 40°C, and three different runs at 10° and 30°C. The S.D. of each data point is smaller than the diameter of the symbol.

Figure 7.

(Top) NAD⁺ synthetase dimer. The A (yellow) and B (green) subunits are represented by ribbons. Key secondary structural elements are labeled; those of the B subunit are primed. The cofactors NaAD, ATP, and Mg⁺² ion are shown as spheres. The twofold symmetry axis is shown as a black line, aligned with the X-axis of the page. All molecular figures created with Ribbons (Carson 1997; see also http://sgce.cbse.uab.edu/ribbons). (Middle) NAD⁺ synthetase dimer interface. The orientation is rotated about 45° about the Y-axis of the image above, to show the maximal amount of surface, and enlarged slightly. The cofactors are shown as ball-and-stick forms, and the A ribbons are drawn smaller to reduce clutter. Only the residues of the A subunit involved in the dimer interface are colored yellow; the rest are now white. Subunit B is green as before. The molecular surface of subunit B within 5 Å of any atom of subunit A is shown colored by atom type assigned for the docking experiments: positive charge, blue; negative charge, red; H-bond donor, cyan; H-bond acceptor, orange; polar, magenta; hydrophobic, green. (Bottom) DMSO docking at the NAD⁺ synthetase dimer interface. The orientation and style is that of the middle panel, but enlarged. Only the residues of the A subunit involved in the dimer interface (yellow) are shown in the ribbon. The surface is shown as a mesh. The DMSO molecules docked to the dimer in the computer simulation are shown as gold spheres.

Figure 8.

Screenshot from a custom SGI Inventor program to “pull apart” the dimer. The subunits are shown as green and yellow ribbons. The substrate associated with the yellow subunit is shown in ball-and-stick form with the Mg⁺² as a silver sphere. The contact surfaces on each subunit (within 5 Å of the other subunit) are shown colored by atomic chemical property and are color coded as shown in Fig. 7 ▶ except for hydrophobic residues, which are shown in white. The interior (backfacing) surface is deep blue. The purple line marks the twofold axis of the dimer (not in the plane of the image.) The cyan line marks the "pull apart" path, perpendicular to the twofold (magenta line).