Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry

Abstract

Nano-electrospray ionization time-of-flight mass spectrometry (ESI-MS) was used to study the conformational consequences of metal ion binding to the colicin E9 endonuclease (E9 DNase) by taking advantage of the unique capability of ESI-MS to allow simultaneous assessment of conformational heterogeneity and metal ion binding. Alterations of charge state distributions on metal ion binding/release were correlated with spectral changes observed in far- and near-UV circular dichroism (CD) and intrinsic tryptophan fluorescence. In addition, hydrogen/deuterium (H/D) exchange experiments were used to probe structural integrity. The present study shows that ESI-MS is sensitive to changes of the thermodynamic stability of E9 DNase as a result of metal ion binding/release in a manner consistent with that deduced from proteolysis and calorimetric experiments. Interestingly, acid-induced release of the metal ion from the E9 DNase causes dramatic conformational instability associated with a loss of fixed tertiary structure, but secondary structure is retained. Furthermore, ESI-MS enabled the direct observation of the noncovalent protein complex of E9 DNase bound to its cognate immunity protein Im9 in the presence and absence of Zn(2+). Gas-phase dissociation experiments of the deuterium-labeled binary and ternary complexes revealed that metal ion binding, not Im9, results in a dramatic exchange protection of E9 DNase in the complex. In addition, our metal ion binding studies and gas-phase dissociation experiments of the ternary E9 DNase-Zn(2+)-Im9 complex have provided further evidence that electrostatic interactions govern the gas phase ion stability.

Fig. 1.

X-ray structure of the E9 DNase (dark) bound to Im9 (light) (Brookhaven Protein accession code 1bxi). The His residues H102, H127, and H131 coordinating the metal ion are shown in black. The phosphate ion located at the active site is labeled. The tryptophan residues W22 and W58 of the E9 DNase are indicated, as well as W74 of Im9. The figure was constructed with WebLab ViewerPro.

Fig. 2.

Nano-electrospray ionization (ESI) mass spectra of the apo-E9 DNase recorded at different pH values: (a) 7.2, (b) 5.3, and (c) 4.1. The ion series labeled with an * has an additional mass of 98 Da.

Fig. 3.

pH-induced unfolding curves for the E9 DNase were deduced by plotting the peak area ratios A_F/(A_U + A_F) as a function of pH. Unfolding curves of holo-E9 DNase and apo-E9 DNase are labeled with (•) and (♦), respectively. Curve indicated with (▴) is deduced from A_{F Zn} /(A_{F Zn} + A_{F apo} + A_U); whereby (F) represents the folded conformer (from 9+ to 7+) and (U) the unfolded conformer (from 10+ to 22+) as illustrated in Figure 2 ▶. The data points for the unfolding curve of holo-E9 DNase were extracted from nano-ESI mass spectra of E9 DNase in the presence of an equimolar amount of Zn²⁺ in 50 mM ammonium acetate adjusted to different pH values. Some representative mass spectra are shown in Figure 6 ▶.

Fig. 4.

Nano-ESI mass spectra of E9 DNase in the presence of increasing amounts of Zn²⁺. Molar ratio of zinc acetate to apo-E9 DNase: (a) 0.25 : 1, (b) 1 : 1, and (c) 4 : 1. Ion peaks of Zn²⁺-bound and metal-free E9 DNase are labeled with filled circles (•) and open circles (○), respectively. The ion series labeled with an * has an additional mass of 98 Da.

Fig. 5.

The relative binding affinities of the E9 DNase for different divalent metal ions. Bars shown in white represent the nonmetal-containing unfolded conformer; bars shown in grey represent the nonmetal-containing folded conformer; and bars shown in black represent the folded metal-containing conformer.

Fig. 6.

Nano-ESI mass spectra of E9 DNase in the presence of an equimolar amount of Zn²⁺ at pH 7.2 (a), 5.3 (b), and 4.1 (c) in 50 mM ammonium acetate. The filled circles (•) represent ion peaks of the Zn²⁺ bound; the open circles (○) represent ion peaks of the metal-free E9 DNase. The repeatedly observed ion series with an additional mass of 98 Da is labeled with an *.

Fig. 7.

Nano-ESI mass spectra of the E9 DNase-Im9-Zn complex in 50 mM ammonium acetate at pH 7.2 at cone voltages of 40 V (a) and 60 V (b). The filled circles (•) represent ion peaks of the E9 DNase containing the Zn²⁺ ion, and open circles (○) correspond to ion peaks of Im9 after cone voltage dissociation. The filled and open circles combined (○•) denote ion peaks representing the E9 DNase-Im9-Zn complex. The insert shows the 5+-ion peak of Im9 after cone voltage dissociation with up to four Na⁺ ions remaining. The ion series labeled with (x) is lacking a mass of 475 ± 3 Da, probably because of a truncated Im9 variant missing the C-terminal tetrapeptide FKQG.

Fig. 8.

Changes in the tryptophan fluorescence emission spectra during acid-induced unfolding of E9 DNase (6 μM), Im9 (6 μM), and E9 DNase-Im9 complex (6 μM) in 50 mM ammonium acetate. (a) Curves 1–5 represent the apo-E9 DNase at pH 7.4, 5.9, 5.3, 4.9, and 3.9; (b) curves 1–5 represent E9 DNase in the presence of a sixfold molar excess of Zn²⁺ at pH 7.4, 5.9, 5.3, 4.9, and 3.9, respectively.

Fig. 9.

Far-UV (a, b) and near-UV (c) CD spectra of E9 DNase in 50 mM ammonium acetate. (a,b) Curves 1–4 represent pH 7.4, 5.9, 5.3, and 3.9 of the apo- and holo E9 DNase (8 μM), respectively. (c) Curves 1 and 2 denote the E9 DNase (50 μM) at pH 7.4 in the absence and presence of Zn²⁺. Curves 3 and 4 represent the E9 DNase at pH 3.9 in the absence and presence of Zn²⁺.