The structure of the proline utilization a proline dehydrogenase domain inactivated by N-propargylglycine provides insight into conformational changes induced by substrate binding and flavin reduction

Abstract

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to its enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the epsilon-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline-reduced, membrane-associated form. The 2.15 A resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane-binding domain include major reorganization of the flavin ribityl chain, severe (35 degrees ) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5) atom, Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the beta1-alphal loop to complete the active site.

Figure 1

Spectroscopic analysis of the inactivation of full-length E. coli PutA by PPG. (A) Flavin spectral changes of E. coli PutA induced by inactivation by PPG. The spectrum of oxidized PutA is represented as the thick curve and has maxima at λ = 451 nm and 378 nm. Spectra were acquired immediately after adding 500 μM PPG and were continually collected for up to 90 min as described in Experimental Procedures. For clarity, only 14 spectra are shown, with the dashed curve indicating the last spectrum. Note that the maximum at λ = 451 nm disappears (decreases) and a new maximum centered at λ = 378 nm appears (increases) as time advances. The inset shows the absorbance recorded at 451 nm (solid circles) and 378 nm (open circles) during the 90-min inactivation process. (B) Denaturation of PPG-inactivated PutA. Spectra are: PutA (7.35 μM) before inactivation (solid curve), PutA inactivated with 500 μM PPG for 90 min (dashed curved), and the flavin species isolated after treatment of PPG-inactivated PutA with 0.2 % SDS (dotted curve).

Figure 2

Kinetics of PutA inactivation by PPG at 25 °C using a dichlorophenolindophenol-based activity assay. The percent activity remaining after incubation with PPG is plotted as a function of time for four inactivator concentrations (0.25 - 2.5 mM). The inset shows the replot of the half-life of inactivation as a function of the reciprocal concentration of PPG. The inactivation parameters obtained from fitting are k_inact = 0.13 ± 0.05 min⁻¹ and K_I = 1.5 ± 0.2 mM.

Figure 3

The two views of PPG-inactivated PutA86-630. Strands and helices are colored pink and blue, respectively. The dashed curves denote disordered segments of the polypeptide chain. The covalent FAD-Lys329 adduct is colored green. Red coloring denotes regions displaying large conformational differences from other PutA PRODH domain structures (i.e., α8 and the β1-α1 loop).

Figure 4

Electron density maps showing the (A) overall FAD conformation and covalent attachment to Lys329 and (B) curvature of the isoalloxazine ring. The cage represents a simulated annealing σ_A-weighted F_o - F_c map contoured at 3σ. Prior to calculation of the map, the FAD and side chain of Lys329 were omitted, and simulated annealing refinement was performed with PHENIX.

Figure 5

Conformational changes during PutA inactivation by PPG detected by limited proteolysis. Purified PutA (1 mg/mL) was incubated for 5 min each with proline (5 mM), THFA (5 mM), or for 30 min with PPG (5 mM), in 50 mM potassium phosphate (pH 7.5) followed by digestion with chymotrypsin (10 μg/mL) for 1 h at 23 °C. The reactions were quenched with phenylmethylsulfonyl fluoride and hot SDS sample buffer after 1 h. After complete denaturation in SDS buffer, 7.5 μg of each digestion was loaded onto an 8 % polyacrylamide denaturing gel for analysis. As a control, undigested ligand-free PutA was also loaded (2.5 μg). The gel was stained with Coomassie Blue to visualize the major products. A molecular mass marker (M) was loaded in the first lane.

Figure 6

Physical binding of PPG-inactivated PutA to E. coli polar lipids. (A) PutA (0.25 mg/ml) was pre-incubated with HEPES-N buffer plus 5 mM proline, 5 mM THFA or 5 mM PPG for 5 min, 5 min or 30 min respectively, and then incubated with freshly prepared E. coli polar lipids (0.8 mg/ml) for 1 hr at room temperature. The soluble and lipid fractions were then separated by Air-fuge ultracentrifugation, denatured with SDS buffer, and analyzed with SDS-PAGE. As a control, an equal amount of denatured PutA without treatment was also loaded. (B) Quantitative analysis of PutA bands for the soluble (gray bars) and lipid (grey hatched bars) fractions normalized as a percentage of total PutA using a Bio-Rad 2000 system.

Figure 7

The FAD conformations observed in three crystal structures of the E. coli PutA PRODH domain: (A) PPG-inactivated PutA86-630, (B) oxidized PutA86-669 complexed with THFA (cyan), and (C) dithionite-reduced PutA86-669. Helix α8 is shown in red for the latter two enzymes; this helix is disordered in PPG-inactivated PutA86-630. Note that the reduced flavins are colored green (panels A, C), whereas the oxidized flavin is colored yellow (panel A). The three flavins are oriented such that the AMP moieties are superimposed.

Figure 8

Close-up view of structural differences between PPG-inactivated PutA86-630 (green) and PutA86-669 complexed with THFA (white), highlighting the movements of Arg431 and Asp370. The occupancy values for the two conformations of Asp370 in the inactivated enzyme are given in parentheses. Orange dashed lines represent hydrogen bonds in the PPG-inactivated enzyme. Black dashed lines represent hydrogen bonds of the THFA complex. The cage represents a simulated annealing σ_A-weighted F_o - F_c map contoured at 3σ. Prior to calculation of the map, the side chains of Asp370 and Arg431 were omitted, and simulated annealing refinement was performed with PHENIX.

Figure 9

Stereographic view of a superposition of the active sites of PPG-inactivated PutA86-630 (green) and PutA86-669 complexed with THFA (white). The THFA ligand is colored cyan. The thin dashed lines indicate hydrogen bonds of the THFA complex. The thick dashed curve denotes the disordered section of α8 of the PPG-inactivated enzyme. For clarity, conformation B of Asp370 of the inactivated enzyme has been omitted.

Figure 10

Stereographic view of a comparison of PPG-inactivated PutA86-630 (green), PutA86-669 complexed with THFA (white), and TtPRODH (orange). The THFA ligand is colored cyan. The thin dashed lines indicate the Arg555-Glu289 ion pair observed in the THFA complex. Note that Glu289 is disordered in the PPG-inactivated enzyme (modeled as Ala), and the corresponding residue of TtPRODH (Glu65) points out of the active site and into the solvent. The thick dashed curve denotes the disordered section of α8 of the PPG-inactivated enzyme.

Scheme 1

Scheme 2

Scheme 3