Regulation of PutA-membrane associations by flavin adenine dinucleotide reduction

Abstract

Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4-electron oxidation of proline to glutamate. Previously, proline was shown to induce PutA-membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA-membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA-membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (K(D)) of <0.01 nM (pH 7.4) for the reduced PutA-lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with K(D) values ranging from approximately 3.6 to 34 nM (pH 7.4) for the PutA-lipid complex. The greater PutA-membrane binding affinity (>300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA-membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA-membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA, confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference ( approximately 24 kJ/mol) between PutA-membrane and PutA-DNA binding.

Figure 1

SPR sensorgrams showing proline and ligand induced PutA-lipid binding. Panel A: Sensorgrams of PutA (20 nM) in the oxidized state (a) and in the presence of 5 mM proline (b) binding to a lipid bilayer of E. coli lipid polar extracts. The arrows indicate the beginning and end of the protein sample injection. In sensorgram a, the rapid change in response units (RU) at the injection start point is due to a refractive index change of the sample, not by protein binding to the lipid bilayer. The RU returned to the initial value after the injection of the sample was complete. Panel B: Oxidized PutA (80 nM) in the presence of 5 mM L-THFA ( ○ ), 5 mM L-lactate ( □ ) and 5 mM DL-P5C ( ◇ ) was injected at 60 μl/min for 120 s onto a L1 chip coated with E. coli polar lipid vesicles. The dissociation phase was observed by the flow of HEPES-N buffer at 60 μl/min for 300 s.

Figure 2

SPR sensorgrams of the association and dissociation kinetics of proline and sodium dithionite reduced PutA-lipid binding. Panel A: From bottom to top, increasing concentrations of PutA (2.5, 5, 10, 20, and 40 nM) in the presence of 5 mM proline (HEPES-N buffer, pH 7.4) were injected onto a L1 chip coated with E. coli polar lipid vesicles. Panel B: From bottom to top, increasing concentrations of PutA (2.5, 5, 10, 20, and 40 nM) in the presence of 10 mM sodium dithionite (HEPES-N buffer, pH 7.4) were injected onto a L1 chip coated with E. coli polar lipid vesicles. Association phase (a) corresponds to the injection of PutA at 60 μl/min for 120 s and the dissociation phase (b) corresponds to the flow of HEPES-N buffer at 60 μl/min for 300 s. The data were fit by global analysis to a 1:1 Langmuir binding isotherm. Signals from the control surface have been subtracted.

Figure 3

Sensorgrams of PutA binding to positively charged lipid vesicles. Panel A: Sensorgrams of proline (5 mM) reduced PutA (10, 20, 40, 80, 160 nM, bottom to top) injected onto a L1 chip coated with EDOPC lipid vesicles. Panel B: Sensorgrams of oxidized PutA (20, 40, 80, 160, 320 nM, bottom to top) injected onto a L1 chip coated with EDOPC lipid vesicles. For all sensorgrams, the association phase (a) corresponds to the injection of the PutA sample at 60 μl/min for 120 s and the dissociation phase (b) corresponds to the flow of HEPES-N buffer at 60 μl/min for 300 s. The data were fit by global analysis to a 1:1 Langmuir binding isotherm. Signals from the control surface have been subtracted.

Figure 4

Effects of DNA binding on PutA membrane associations. Panel A: Oxidized PutA (100 nM) in the presence of sodium lactate (5 mM) was injected onto a L1 chip coated with E. coli polar lipid vesicles (arrows 1 and 2). The lipid-bound PutA was then washed with HEPES-N buffer at 60 μl/min for 180 s (arrows 2-3) followed by injection of put control DNA (419 bp, 20 nM) (arrows 3-4). The rapid change in RU at the beginning of the injection of DNA is due to the refractive index change of DNA sample, not to PutA-DNA binding. The RU returned to the initial value after the injection of the DNA sample was complete. Panel B: Sensorgrams of the PutA (10 nM)-oligonucleotide (1.8 μM) complex (upper trace) and the PutA (10 nM)-put control DNA (300 nM) complex (lower trace) injected onto a L1 chip coated with E. coli polar lipid vesicles in the presence of 5 mM proline (arrows 1 and 2).

Figure 5

Gel mobility shift assay of the oligonucleotide binding site complexed with PutA. IRdye-700 labeled 21-bp duplex DNA (5 nM) and varying concentrations of PutA (0-400 nM dimer) were incubated in binding mixtures (HEPES-N buffer, pH 7.4) containing 100 μg/ml of nonspecific calf thymus DNA at 20 °C for 20 min. The complexes were separated using a nondenaturing polyacrylamide gel (8%).

Figure 6

Thermodynamic model for how FAD reduction and ligand binding regulate PutA intracellular location and function (transcriptional repressor vs. membrane associated enzyme). Free energy of PutA-DNA binding and midpoint potential (E_m) values are from reference () which were determined in 70 mM Tris buffer (pH 7.5) at 20 °C.