Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

<div><p>The cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.</p> </div

Thermodynamic parameters of WT human c-NADP-ME and the interface mutants during urea-induced denaturation.

a<p>These data were derived from fitting the results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050143#pone-0050143-g002" target="_blank">Figure 2</a> and were monitored using circular dichroism spectrometry.</p

Midpoints of ANS-fluorescence and half inactivation for WT human c-NADP-ME and the interface mutants during urea-induced denaturation.

a<p>These data were derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050143#pone-0050143-g003" target="_blank">Figure 3</a> and were monitored using ANS-fluorescence.</p>b<p>These data were derived from the fitting results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050143#pone-0050143-g004" target="_blank">Figure 4</a> and were monitored using an activity assay.</p

Continuous sedimentation coefficient distributions of WT c-NADP-ME and the interface mutants during urea denaturation.

<p>The enzymes were preincubated in various urea concentrations in 30 mM Tris-acetate (pH 7.4) at 25°C for 16 h and then run in an analytical ultracentrifuge at 20°C. <b>Panels A–G:</b> WT. <b>Panels H–N:</b> H142A/D568A. <b>Panels O–U:</b> H51A/D90A.</p

The urea-induced unfolding profiles of WT c-NADP-ME and interface mutants, as monitored using CD spectrometry.

<p>The enzymes were preincubated in various urea concentrations in 30 mM Tris-acetate (pH 7.4) at 25°C for 16 h. <b>A.</b> WT. <b>B.</b> W572A. <b>C.</b> H142A. <b>D.</b> H142/D568A. <b>E.</b> H51A/D90A. <b>F.</b> H51A/D139A. The experimental data are the mean residue ellipticity at 222 nm ((Θ)₂₂₂) monitored by far-UV CD. All data were fitted using a three-state model. The fitting results and residues are displayed as a solid line with error bars.</p

Dimer and tetramer interfaces of cytosolic malic enzyme.

<p>The dimer of dimers quaternary structure of cytosolic malic enzyme (PDB code: 1GQ2). The amino acid residues at the dimer interface, His 51, Asp 90, Asp 139, His 142, Asp 568 and Trp 572 in the dimer and tetramer interface are represented by ball-and-stick modeling. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).</p

Urea-induced enzyme inactivation of WT human c-NADP-ME and the interface mutants.

<p>The enzymes were preincubated in various urea concentrations in 30 mM Tris-acetate (pH 7.4) at 25°C for 16 h and then monitored by following NADPH production at an absorbance of 340 nm. All data were fitted using a two-state model, and the fitting results and residues are displayed as a solid line with error bars.</p

Inhibitory effect of ATP on human WT and mutant c-NADP-ME.

<p>The inhibited enzyme activities were assayed with NAD⁺ (<b>A</b>) or NADP⁺ (<b>B</b>) as the cofactor. The assay mixture contains 40 mM malate, 10 mM MgCl₂, and 1 mM NAD⁺ or NADP⁺. The ATP concentration ranged from 0 to 3 mM.</p

Specificity constants for human wild-type and nucleotide-binding mutant c-NADP-ME variants.

<p>Specificity constants for human wild-type and nucleotide-binding mutant c-NADP-ME variants.</p

Kinetic parameters for human wild-type and nucleotide-binding mutant variants of c-NADP-ME.

<p>Kinetic parameters for human wild-type and nucleotide-binding mutant variants of c-NADP-ME.</p