Biochemical and biophysical investigations of the interaction between human glucokinase and pro-apoptotic BAD

<div><p>The glycolytic enzyme glucokinase (GCK) and the pro-apoptotic protein BAD reportedly reside within a five-membered complex that localizes to the mitochondria of mammalian hepatocytes and pancreatic β-cells. Photochemical crosslinking studies using a synthetic analog of BAD’s BH3 domain and <i>in vitro</i> transcription/translation experiments support a direct interaction between BAD and GCK. To investigate the biochemical and biophysical consequences of the BAD:GCK interaction, we developed a method for the production of recombinant human BAD. Consistent with published reports, recombinant BAD displays high affinity for Bcl-xL (K_D = 7 nM), and phosphorylation of BAD at S118, within the BH3 domain, abolishes this interaction. Unexpectedly, we do not detect association of recombinant, full-length BAD with recombinant human pancreatic GCK over a range of protein concentrations using various biochemical methods including size-exclusion chromatography, chemical cross-linking, analytical ultracentrifugation, and isothermal titration calorimetry. Furthermore, fluorescence polarization assays and isothermal titration calorimetry detect no direct interaction between GCK and BAD BH3 peptides. Kinetic characterization of GCK in the presence of high concentrations of recombinant BAD show modest (<15%) increases in GCK activity, observable only at glucose concentrations well below the <i>K</i>_0.5 value. GCK activity is unaffected by BAD BH3 peptides. These results raise questions as to the mechanism of action of stapled peptide analogs modeled after the BAD BH3 domain, which reportedly enhance the <i>V</i>_max value of GCK and stimulate insulin release in BAD-deficient islets. Based on our results, we postulate that the BAD:GCK interaction, and any resultant regulatory effect(s) upon GCK activity, requires the participation of additional members of the mitochondrial complex.</p></div

Development of a protocol for production and purification of recombinant BAD-GST.

<p>(A) Overview of the optimized protocol for BAD-GST production. (B) Impact of refolding buffer additives upon BAD-GST aggregation, as evaluated by the spin-filter binding assay developed by Bondos [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171587#pone.0171587.ref025" target="_blank">25</a>]. (C) Size-exclusion chromatogram of HisTrap purified BAD-GST in the absence (gray) and presence (black) of arginine (0.5 M) and CHAPS (2.5 mM). (D) Coomassie blue stained SDS-PAGE of BAD-GST throughout the purification protocol. <i>Lane 1</i>: Insoluble fraction following cell lysis; <i>Lane 2</i>: Soluble fraction; <i>Lane 3</i>: Ni-NTA affinity column flow through; <i>Lane 4</i>: Ni-NTA affinity column eluate; <i>Lane 5</i>: glutathione-affinity column flow through; <i>Lane 6</i>: glutathione-affinity column eluate; <i>Lane 7</i>: Size-exclusion purified BAD-GST.</p

Characterization of recombinant BAD-GST.

<p>(A) CD spectra of recombinant BAD-GST (black), BAD following cleavage and removal of the GST tag (gray), BAD-GST following <i>in vitro</i> phosphorylation with PKAc (purple) and GST (green). (B) Analytical ultracentrifugation sedimentation velocity analysis of BAD-GST (black), GST (green), Bcl-xL (blue) and a 1:1 molar mixture of BAD-GST with Bcl-xL (red). (C) Size-exclusion chromatogram of BAD-GST (black), BAD following cleavage and removal of the GST tag (gray), and GST (green).</p

Characterization of the interaction between GCK and peptide analogs of the BAD BH3 domain.

<p>(A) GCK at concentrations up to 10 μM does not increase the fluorescence polarization of f-BH3; (B) Isotherms for the addition of phosphorylated BH3 peptide to GCK do not indicate an interaction.</p

Functional assays of recombinant BAD-GST.

<p>(A) Representative isotherm for the interaction of BAD-GST and Bcl-xL. The thermodynamic parameters represent the average of two independent experiments. (B) Size exclusion chromatogram of BAD-GST (black), Bcl-xL (blue) and a 1:1 mixture of BAD-GST and Bcl-xL (red). Coomassie blue stained SDS-PAGE analysis of individual fractions (above), confirms formation of a higher molecular weight complex between BAD-GST and Bcl-xL. (C) Fluorescence polarization assays demonstrate that f-BH3 binding to Bcl-xL (top) is disrupted by recombinant BAD-GST (black) or BAD following cleavage and removal of the GST tag (gray), with IC₅₀ values of 130 nM and 440 nM, respectively. Phosphorylation of BAD-GST by PKA (pink) abrogrates the protein’s ability to compete with f-BH3. Phosphomimetics demonstrate that phosphorylation at S118 (red), but not S75 and S99 (green), is responsible for impaired binding to Bcl-xL.</p