Flavodoxin cofactor binding induces structural changes that are required for protein-protein interactions with NADP(+) oxidoreductase and pyruvate formate-lyase activating enzyme

Abstract

Flavodoxin (Fld) conformational changes, thermal stability, and cofactor binding were studied using circular dichroism (CD), isothermal titration calorimetry (ITC), and limited proteolysis. Thermodynamics of apo and holo-Fld folding were examined to discern the features of this important electron transfer protein and to provide data on apo-Fld. With the exception of fluorescence and UV-vis binding experiments with its cofactor flavin mononucleotide (FMN), apo-Fld is almost completely uncharacterized in Escherichia coli. Fld is more structured when the FMN cofactor is bound; the association is tight and driven by enthalpy of binding. Surface plasmon resonance binding experiments were carried out under anaerobic conditions for both apo- and holo-Fld and demonstrate the importance of structure and conformation for the interaction with binding partners. Holo-Fld is capable of associating with NADP(+)-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. Limited proteolysis experiments were analyzed by LC-MS to identify the regions in Fld that are involved in conformation changes upon cofactor binding. Docking software was used to model the Fld/PFL-AE complex to understand the interactions between these two proteins and gain insight into electron transfer reactions from Fld to PFL-AE.

Keywords: Circular dichroism (CD); Flavodoxin; NADP(+) oxidoreductase; Protein–protein interactions; Pyruvate formate-lyase activating enzyme; Surface plasmon resonance (SPR).

Figure 1

Secondary Structure of E. coli Fld as determined by far-UV CD. Black: Native apo-Fld at 24 °C, Blue: Native holo-Fld at 24 °C, Red: Unfolded apo-Fld at 86 °C, Green: Unfolded holo-Fld at 94 °C. Protein concentrations were 30 μM, 0.1 mm cuvette, 195-260 nm, Sensitivity = 100 millidegrees, data pitch = 0.1 nm, accumulation of 4 scans.

Figure 2

Thermal unfolding curves for holo-Fld as monitored in the near-UV, and far-UV regions by CD. A) Apo-Fld near-UV (▲) and far-UV (●) unfolding curves. B) Holo-Fld near-UV (■) and far-UV (▼) unfolding curves. The unfolding curves were globally fit to a three state folding model.

Figure 3

Isothermal titration calorimetry of apo-Fld binding FMN at 37 °C. Injections (26 at 10 μL each and the first one at 2 μL) of FMN (600 μM stock concentration) were added to 25 μM apo-Fld in the cell. The reference power was 10 μcal/second, initial delay of 300 seconds, stir speed of 510 rpm, feedback mode/gain was set to high and automatic with fast equilibration, duration of 24 seconds, spacing of 300 seconds, with a filter period of 2 seconds

Figure 4

Binding of holo-Fld to FNR as monitored by CD spectroscopy. Blue: 30 μM FNR; Green: 30 μM Fld; Red: spectral addition of FNR and Fld; Black: Mixture of 30 μM FNR and 30 μM Fld. Inset shows difference spectra corresponding to a titration of (0 μM, 7.5 μM, 15 μM, 22.5 μM, 30 μM, 60 μM) Fld into FNR (30 μM) to show changes in the near-UV and visible regions as a result of Fld binding FNR.

Figure 5

Binding of PFL-AE to apo- and holo-Fld using surface plasmon resonance under anaerobic conditions. Single cycle kinetics mode was used at 25 °C and PFL-AE was covalently attached to a CM5 sensor chip using thiol coupling. Either apo-Fld (●) or holo-Fld (■) was titrated over PFL-AE at concentrations of 2.47, 7.41, 22.2, 66.7, and 200 μM. The running buffer used was 20 mM HEPES, 10 mM KCl, pH 7.4 with a flow rate of 30 μL/minute used in assays with a contact time of 60 seconds and dissociation time of 120 seconds. The regeneration buffer was 20 mM HEPES, 500 mM KCl, 0.005 % Polysorbate 20, 200 mM imidazole, pH 7.4 and the regeneration time was 120 seconds.

Figure 6

Conformational changes in Fld induced by FMN cofactor binding. Apo- and holo-Fld was digested with trypsin and peptides were analyzed by LC-MS. Blue: 90's loop of Fld connected to β-sheet 4, Red: 50's loop of Fld connected to α-helix 3. The side chain is shown for the unique apo-Fld cleavage point at K81 and it is colored yellow and labeled. The FMN cofactor is colored orange.

Figure 7

A) Surface representation of PFL-AE with 100 % sequence conserved residues colored red. B) Docked structure of Fld bound to PFL-AE constructed using ZDOCK: Fld (1AHN), PFL-AE (3C8F). C) Zoomed in view of the active sites in the Fld/PFL-AE complex. The side chains are colored red for aromatic residues that form a bridge between cofactors. The FMN cofactor, W57 from flavodoxin, W42 from PFL-AE, and the [4Fe-4S] cluster are all labeled.