Potassium and Ionic Strength Effects on the Conformational and Thermal Stability of Two Aldehyde Dehydrogenases Reveal Structural and Functional Roles of K⁺-Binding Sites

<div><p>Many aldehyde dehydrogenases (ALDHs) have potential potassium-binding sites of as yet unknown structural or functional roles. To explore possible K⁺-specific effects, we performed comparative structural studies on the tetrameric betaine aldehyde dehydrogenase from <em>Pseudomonas aeruginosa</em> (PaBADH) and on the dimeric BADH from spinach (SoBADH), whose activities are K⁺-dependent and K⁺-independent, respectively, although both enzymes contain potassium-binding sites. Size exclusion chromatography, dynamic light scattering, far- and near-UV circular dichroism, and extrinsic fluorescence results indicated that in the absence of K⁺ ions and at very low ionic strength, PaBADH remained tetrameric but its tertiary structure was significantly altered, accounting for its inactivation, whereas SoBADH formed tetramers that maintained the native tertiary structure. The recovery of PaBADH native tertiary-structure was hyperbolically dependent on KCl concentration, indicating potassium-specific structuring effects probably arising from binding to a central-cavity site present in PaBADH but not in SoBADH. K⁺ ions stabilized the native structure of both enzymes against thermal denaturation more than did tetraethylammonium (TEA⁺) ions. This indicated specific effects of potassium on both enzymes, particularly on PaBADH whose apparent <em>T</em>_m values showed hyperbolical dependence on potassium concentration, similar to that observed with the tertiary structure changes. Interestingly, we also found that thermal denaturation of both enzymes performed in low ionic-strength buffers led to formation of heat-resistant, inactive soluble aggregates that retain 80% secondary structure, have increased β-sheet content and bind thioflavin T. These structured aggregates underwent further thermal-induced aggregation and precipitation when the concentrations of KCl or TEACl were raised. Given that PaBADH and SoBADH belong to different ALDH families and differ not only in amino acid composition but also in association state and surface electrostatic potential, the formation of this kind of β-sheet pre-fibrillar aggregates, not described before for any ALDH enzyme, appear to be a property of the ALDH fold.</p> </div

DLS data of PaBADH and SoBADH under different salt conditions.

a<p>The mean radius corresponds to the highest peak of the size distribution histograms shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054899#pone-0054899-g001" target="_blank">Figure 1</a>.</p>b<p>Calculated using the program HYDROPRO (version 10) and the atomic coordinates of PaBADH (PDB accession code 2WME) or SoBADH (PDB accession code 4A0M).</p

Effects of the concentration of monovalent cations on the thermal denaturation profiles of PaBADH.

<p>Temperature-induced changes in CD ellipticity at 222 nm were measured at increasing concentrations of cations. The thermal profiles shown have been shifted 10 mdeg over the <i>y-</i>axis for clarity of the figure. From bottom to top: Enzyme samples (0.25 mg/ml) incubated in the non-salt buffer (open squares) or in the presence of 25, 37.5, 50, 150 and 250 mM KCl <b>(A),</b> or of 25, 50, 75, 100 and 250 mM TEACl (<b>B</b>). The thermal profile at zero salt starts at –27.01 mdeg and ends at –20.37 mdeg. Changes in the temperature of thermal transition (solid squares) at increasing KCl <b>(C)</b> or TEACl concentrations (<b>D</b>). In (<b>C</b>) and (<b>D</b>) the <i>T</i>_m values of the second thermal transitions (solid triangles) observed at 37.5 and 50 mM KCl or at 50 and 75 mM TEACl are also shown. The temperature range was 20–90°C and the scan rate was 1.5°C/min. The solid lines in <b>(A)</b> and <b>(B)</b> represent the best fit of the single or double thermal transition data to a single or double sigmoidal Boltztmann functions, respectively, by non-linear regression analysis.</p

K⁺-binding sites of PaBADH.

<p>(<b>A</b>) The intra-subunit cation-binding site of subunit A. Similar sites exist in the other three subunits. (<b>B</b>) The inter-subunit cation-binding site observed between subunits A and B. Another symmetrical site exists between these two subunits, and there are two more between subunits C and D. (<b>C</b>) Central-cavity cation-binding site observed between subunits A, C and D. For clarity of the figure subunit B, which do not participate in this site, is not shown. Similar sites are present between subunits B, C and D, between subunits C, B and A, and between subunits D, B and A. K⁺ ions are shown as violet balls and water molecules as smaller red balls; amino acid side-chains are depicted as sticks with carbon atoms colored according to the subunit (grey for subunit A, yellow for B, pink for C, and green for D), oxygen in red and nitrogen in blue. Ion coordination bonds are depicted as black long-dashed lines. Images were generated using the PyMOL (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>) and the PaBADH coordinates from the crystal of PDB accession code 2WME.</p

PaBADH conformational changes induced by the progressive increase in KCl concentration in the incubation medium.

<p>Enzyme samples (0.25 mg/ml) in non-salt buffer were incubated at 20°C with increasing concentrations of KCl; after 3 min equilibration, the near-UV CD spectra and the fluorescence emission spectra of ANS were recorded. Changes in the ellipticity value at 280 nm of the near UV-CD spectra (open circles) and in fluorescence intensity at 482 nm in the emission spectra of ANS (closed circles) are plotted as a function of KCl concentration. The points represent the absolute value of the observed changes and the curves correspond to the fit of the data to the Hill equation by nonlinear regression.</p

Effect of heating at low ionic strength on the secondary structure^a of PaBADH and SoBADH^b.

a<p>Estimated using the deconvolution software CDPro <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054899#pone.0054899-Sreerama1" target="_blank">[28]</a>.</p>b<p>SoBADH values are given in parenthesis.</p

Secondary and tertiary structural changes induced by temperature at low ionic strength on PaBADH.

<p>The thermal denaturation of the enzyme (0.25 mg/ml) in the non-salt buffer was monitored by changes in ellipticity in the far-UV CD at 222 nm (closed triangles), near-UV CD at 282 nm (closed circles), and ANS fluorescence emission intensity at 482 nm (closed squares). The temperature range was 20–90°C and the scan rate 1.5°C/min. The CD signals are represented as fraction of the values observed at 20°C. The ANS signal was normalized using the maximum fluorescence intensity observed at 42°C.</p

Effect of salts on the thermal denaturation of PaBADH and SoBADH.

<p>Heat-induced transitions of PaBADH (<b>A</b>) and SoBADH (<b>B</b>) samples (0.25 mg/ml) in the non-salt buffer (squares), K-buffer (open triangles) or TEA-buffer (open circles) were determined by following the changes at 222 nm in the far-UV CD signal. The temperature range was 20–90°C and the scan rate 1.5°C/min. Transitions were evaluated with a Boltzmann function.</p

Structural properties of PaBADH thermally denatured in the non-salt buffer.

<p>(<b>A</b>) Far-UV CD spectra of heat-denatured protein at 90°C (long-dash lines) and after cooling back to 20°C (solid lines). (<b>B</b>) Near-UV CD spectra of the enzyme after cooling back to 20°C (solid lines), and after denaturation with 6 M guanidinium chloride (dotted line). Enzyme samples (0.25 mg/ml) were heated at a rate of 1.5 min/°C. (<b>C</b>) SEC elution profile of enzyme (0.25 mg/ml) after being heated at 37°C for 2 h (solid line). (<b>D</b>) ThT fluorescence emission spectrum of the sample heated at 90°C and cooled back to 20°C (solid line). With comparative purposes, the CD and ThT fluorescence spectra as well as the SEC elution profile of the same samples obtained at 20°C before heating are shown as dash-dotted lines.</p