Figure 1.
Schematic of OpuA from L. lactis.
Panel A represents the different constructs used in this paper. OpuAA consists of the nucleotide-binding domain and tandem CBS domains connected to the anionic tail (approximately 20 amino acids, indicated in red). OpuABC consists of the transmembrane domain (TMD, OpuAB: I is amphipatic helix; II–VII are transmembrane segments; SA is the signal anchor sequence), connected to the substrate-binding domain (SBD, OpuAC). Panel B shows the organization of the subunits in the membrane. The functional protein is a dimer of the complex shown in panel B. The OpuABC subunits are depicted in grey, the OpuAA subunits in orange.
Figure 2.
Glycine betaine-binding to purified OpuA (panel A & C) or OpuAC (panel B & D).
Binding was measured using intrinsic protein fluorescence (panel A & B), filter-based assay using radio-labeled ligand (panel C), and isothermal titration calorimetry (panel D) at various concentrations of potassium phosphate (10–250 mM KPi, pH 7.0). The intrinsic protein fluorescence measurements and the filter-based assay were done using a concentration of ∼0.5 µM purified OpuAC. The fluorescent measurements were plotted as the absolute percentage change of the initial fluorescence signal in the absence of substrate (panel A & B); the filter-based measurements were corrected for background signals (panel C); and the data fits are shown as black lines. In panel A–C: measurements were done at 250 mM KPi (▪), 50 mM KPi (•) and 10 mM KPi (panel A and C) or 12.5 mM KPi (panel B) (▴). ITC measurements (panel D) were performed at 50 µM of purified OpuAC and the integrated heat peaks were fitted to a one site-binding model (black line, lower panel).
Table 1.
Glycine betaine binding to purified OpuA and OpuAC using intrinsic protein fluorescence.
Figure 3.
Substrate binding specificity of purified OpuAC.
Binding was measured using intrinsic protein fluorescence at an OpuAC concentration of ∼0.5 µM. The following substrates were tested: proline (panel A), carnitine (panel B), proline betaine (panel C) and glycine betaine (Panel D–F). In the panels D–F, OpuAC was pre-incubated with different concentrations of proline (panel D), carnitine (panel E) or proline betaine (Panel F) as inhibitor, prior to titration with glycine betaine. In panel D–F, the concentrations of inhibitor are: no inhibitor (▪), 25 mM proline/carnitine or 0.125 mM proline betaine (•), 50 mM proline/carnitine or 0.25 mM proline betaine (▴), 100 mM proline/carnitine or 0.5 mM proline betaine (▾), 250 mM carnitine or 1 mM proline betaine (⧫) and 500 mM carnitine (★). Measurements in panel D–F were fitted with equation 1 (using a KD of 4 µM for glycine betaine) and the inhibitor concentrations indicated. The inhibition experiments with proline and carnitine show a decrease in the ΔFmax at increased concentrations of inhibitor, which is caused by the high concentrations of inhibitor (see panel A and B). The fluorescence, however, reached the same end level in all measurements, which is indicative for specific and competitive binding.
Table 2.
Binding of various ligands to purified OpuAC using intrinsic protein fluorescence.
Figure 4.
Static light-scattering analysis of purified OpuAC.
Purified OpuAC, in the absence or presence of 1 mM glycine betaine, was run on a gel filtration column, coupled to detectors for UV absorbance, refractive index and light scattering. The molecular mass was calculated throughout the elution peaks.
Figure 5.
X-ray crystallography structure of OpuAC and its binding site.
Panel A shows the structures of OpuAC in its closed (orange) and open (gray) conformations, highlighting the opening of the protein. Panel B, superimposition of the binding pockets for the open and closed-liganded structures of OpuAC. Upon closure of the protein a complete Trp-prism is formed for coordination of the quaternary ammonium moiety of glycine betaine. In addition, hydrogen bonds are formed between the carboxylate of glycine betaine and H392, G437 and V438, thereby stabilizing the closed conformation.
Table 3.
Data collection and refinement statistics.