Allosteric Regulation of Mammalian Pantothenate Kinase

Abstract

Pantothenate kinase is the master regulator of CoA biosynthesis and is feedback-inhibited by acetyl-CoA. Comparison of the human PANK3·acetyl-CoA complex to the structures of PANK3 in four catalytically relevant complexes, 5'-adenylyl-β,γ-imidodiphosphate (AMPPNP)·Mg²⁺, AMPPNP·Mg²⁺·pantothenate, ADP·Mg²⁺·phosphopantothenate, and AMP phosphoramidate (AMPPN)·Mg²⁺, revealed a large conformational change in the dimeric enzyme. The amino-terminal nucleotide binding domain rotates to close the active site, and this allows the P-loop to engage ATP and facilitates required substrate/product interactions at the active site. Biochemical analyses showed that the transition between the inactive and active conformations, as assessed by the binding of either ATP·Mg²⁺ or acyl-CoA to PANK3, is highly cooperative indicating that both protomers move in concert. PANK3(G19V) cannot bind ATP, and biochemical analyses of an engineered PANK3/PANK3(G19V) heterodimer confirmed that the two active sites are functionally coupled. The communication between the two protomers is mediated by an α-helix that interacts with the ATP-binding site at its amino terminus and with the substrate/inhibitor-binding site of the opposite protomer at its carboxyl terminus. The two α-helices within the dimer together with the bound ligands create a ring that stabilizes the assembly in either the active closed conformation or the inactive open conformation. Thus, both active sites of the dimeric mammalian pantothenate kinases coordinately switch between the on and off states in response to intracellular concentrations of ATP and its key negative regulators, acetyl(acyl)-CoA.

Keywords: X-ray crystallography; acetyl coenzyme A (acetyl-CoA); allosteric regulation; coenzyme A (CoA); cooperativity; enzyme kinetics; vitamin.

FIGURE 1.

Catalytic cycle and acyl-CoA inhibition of PANK3. PANK3 operates by a compulsory ordered mechanism with ATP as the leading substrate (7). The PANK3 dimer exists in two distinct conformations. The inactive conformation has an “open” carboxyl-terminal nucleotide binding domain that is stabilized by acetyl-CoA binding (red). The active conformation has a “closed” nucleotide binding domain that is stabilized by ATP binding (green). Acetyl-CoA and ATP binding to PANK3 is competitive (10). Pantothenate binds to the PANK3·ATP·Mg²⁺ complex, and following catalysis, 4-phosphopantothenate is released followed by ADP. 4-Phosphopantothenate is rapidly converted to CoA and its thioesters, which function as feedback regulators of the enzyme. This work describes the structural basis for the highly cooperative transition between the active (ATP-bound) and inactive (acyl-CoA-bound) conformations, and the structural alterations that occur at each intermediate step in the catalytic cycle. The PDB codes for the determined structures are shown next to the model representations. Although ligand-free PANK3 must exist, the structure(s) has not been determined (red/green state).

FIGURE 2.

Comparison of the overall structures of the inactive and active PANK3 conformations. A, inactive open PANK3 conformation stabilized by its interactions with acetyl-CoA that binds in the nucleotide pocket and extends through the substrate-binding site to the dimer interface (PDB code 3MK6). The P-loop is shown in magenta, and acetyl-CoA is shown in cyan. B, active closed PANK3 conformation stabilized by AMPPNP·Mg²⁺ and pantothenate binding. The nucleotide binding pocket is closed with AMPPNP (gray) engaging the P-loop (magenta) and pantothenate (green) in the substrate pocket. The domain closure in the inactive to active transition is mediated by a rotation around the axis of helix α4 (purple). The ∼40° rotation of helix α1 to the closed conformation breaks α1 into two helical segments (α1a and α1b), and the disordered loop at the carboxyl terminus of helix α1 becomes a short β-ribbon that associates with the outside edge of the subdomain's β-sheet.

FIGURE 3.

Substrate/product interactions within the active site during the PANK3 catalytic cycle. A, active site structure of the PANK3·AMPPNP·Mg²⁺ complex (PDB code 5KPT). B, active site structure of the PANK3·AMPPNP·Mg²⁺·pantothenate quaternary complex (PDB code 5KPR). C, active site structure of the PANK3·ADP·Mg²⁺·phosphopantothenate complex (PDB code 5KPZ). D, active site structure of the PANK3·AMPPN·Mg²⁺ complex (PDB code 5KQ8). The active site is composed of residues derived from ATP-binding protomer shown in yellow, and residues contributed by the opposite protomer are shown in cyan. E–H, 2F_o − F_c electron density maps contoured at 1σ for the ligands and the key structured water molecules in the active site complexes. E, AMPPNP·Mg²⁺. F, AMPPNP·Mg²⁺·pantothenate. G, ADP·Mg²⁺·phosphopantothenate. H, AMPPN·Mg²⁺.

FIGURE 4.

Role of Glu-138 in PANK3 catalysis. A, view of the PANK3 active site illustrating the environment of Glu-138 in the PANK3·AMPPNP·Mg²⁺·pantothenate complex. B, purification of PANK3(E138A) by gel filtration chromatography on a calibrated Sepharose S-200 column (black). The elution profile of PANK3 is shown in red. The purity of the final PANK3(E138A) protein was assessed by gel electrophoresis (inset). C, PANK3(E138A) was catalytically inactive. D, PANK3(E138A) was correctly folded based on the concentration-dependent thermal stabilization of the protein by ATP. These data were compiled from two technical replicates from two independent biological experiments, and the line is the data points fitted to the non-linear Michaelis-Menten equation. A representative experiment comparing the thermal stability of PANK3(E138A) in the presence and absence of 8 mm ATP (inset) is shown. The peaks of the first derivative plots of the thermal denaturation curves identify the temperature at which 50% of the protein is unfolded. The average thermal stabilization was calculated from triplicate measurements, rounded to the nearest degree, and shown in the figure panel inset. E, circular dichroism spectra of PANK3, PANK3(E138A), and PANK3(G19V) show that the mutant proteins were structured. F, “road-kill” diagram of the key interactions in the PANK3 active site and the chemical mechanism for the phosphorylation of pantothenate.

FIGURE 5.

Substrate interactions in PANK3. A, x-ray structure of the PANK3·AMPPNP·Mg²⁺·pantothenate complex (Pan) (green) is overlaid with the PANK3·acetyl-CoA complex (gray). Pantothenate and the pantothenate portion of acetyl-CoA superimpose and make the same key interactions with Arg-207 and Ser-195. Hydrogen bonds in the PANK3·AMPNP·Mg²⁺·pantothenate and PANK3·acetyl-CoA structures are shown as red or black dashed lines, respectively. B, representative thermal stabilization profiles displayed as the first derivative of the denaturation curve of PANK3 in the presence of 2 mm AMPPNP·Mg²⁺ (green), 10 mm pantothenate (Pan) (red), pantetheine (PanSH) (blue), and N-heptylpantothenamide (N7-Pan) (black). The peaks of the first derivative plots of the thermal denaturation curves identify the temperature at which 50% of the protein is unfolded. Thermal stabilization was calculated from triplicate measurements, and the average changes in temperature were rounded to the nearest degree, which were 1 °C for pantetheine and 11 °C for N-heptylpantothenamide. C, concentration-dependent thermal stabilization of PANK3 by pantothenate and N-heptylpantothenamide. These data were compiled from two technical replicates from two independent biological experiments, and the line is the data points fitted to the non-linear Michaelis-Menten equation. D, overlay of the PANK3·AMPPNP·Mg²⁺·pantothenate complex (green) and the ADP·N-heptylpantothenamide (PDB code 3SMS) structures (orange). The hydrocarbon tail of the N-heptylpantothenamide substrate reaches across the dimer interface to interact with Trp-341′ on helix α10 on the opposite protomer. Pantothenate does not contact helix α10 and Trp-341′ assumes a different conformation.

FIGURE 6.

Cooperative binding of ATP to PANK3. A, kinetic analysis of PANK3 with respect to the ATP concentration. B, kinetic analysis of PANK3 with respect to the pantothenate concentration. These data were compiled from duplicate technical replicates in two independent biological experiments. The lines are the fit to the Hill equation giving rise to the K_0.5 and Hill number (H) parameters shown in the figure panels. C, representative experiment showing that the addition of 8 mm ATP (black) compared with buffer alone (red) stabilizes PANK3 to thermal denaturation. The peaks of the first derivative plots of the thermal denaturation curve identify the temperature at which 50% of the protein is unfolded. Thermal stabilization was calculated from triplicate technical replicates from two independent experiments, and the average changes in temperatures were rounded to the nearest degree and shown in the figure panels. D, concentration dependence of PANK3 thermal stabilization by ATP·Mg²⁺. These data were compiled from two technical replicates from two independent biological experiments, and the line is the data points fitted to the non-linear Michaelis-Menten equation.

FIGURE 7.

Characterization of PANK3/PANK3(G19V) heterodimers. A, structure of the PANK3·AMPPNP·Mg²⁺ complex showing the location of Gly-19 on the P loop. The mutation of Gly-19 to Val-19 introduces a bulky side chain into the P-loop that interferes with ATP binding. B, structure of the PANK3·acetyl-CoA complex illustrating the location of Gly-19 in this conformation. The P-loop has moved away from the adenine pocket, and the introduction of a bulky valine at this position would not be predicted to interfere with acetyl-CoA binding. C, immunoblotting of the PANK3, PANK3/PANK3(G19V) heterodimers, and PANK3(G19V) with anti-His tag and anti-FLAG tag antibodies. In the heterodimer, the PANK3 subunit was His-tagged and the PANK3(G19V) subunit was FLAG-tagged. PANK3 (WW), PANK3/PANK3(G19V) heterodimers (VW), and PANK3(G19V) (VV) immunoblots and CBB is the Coomassie-stained gel with molecular mass markers are shown. D, enzymatic activity of PANK3 compared with PANK3/PANK3(G19V) heterodimers and PANK3(G19V). E, kinetic analysis of PANK3/PANK3(G19V) heterodimers with respect to ATP illustrating the loss of cooperativity and ATP affinity. F, kinetic analysis of PANK3/PANK3(G19V) heterodimers with respect to pantothenate. The kinetic data were derived from two technical replicates from two independent experiments, and the lines are the data fit to the Hill equation as described under “Experimental Procedures.”

FIGURE 8.

Biochemical analysis of ATP and acetyl-CoA binding to PANK3 and PANK3/PANK3(G19V) heterodimers. The peaks of the first derivative plots of the thermal denaturation curves identify the temperatures at which 50% of the protein is unfolded. Representative thermal denaturation curves are shown. The shifts in the thermal denaturation temperatures were calculated from experiments performed in triplicate, and the averages were rounded to the nearest degree and shown in the figure panels. Samples contained buffer alone (red) or buffer plus 10 mm ATP·Mg²⁺ (black) or 10 μm acetyl-CoA (blue). A, PANK3(G19V) was not stabilized to thermal denaturation by 10 mm ATP·Mg²⁺. B, acetyl-CoA (10 μm) stabilized PANK3(G19V) to thermal denaturation by 8 °C. C, PANK3/PANK3(G19V) heterodimers exhibited two thermal transitions in the presence of 10 mm ATP·Mg²⁺. D, acetyl-CoA (10 μm) stabilizes PANK3/PANK3(G19V) heterodimers to thermal denaturation.

FIGURE 9.

Realignment of the dimer interface simultaneously alters the conformation of both protomers. View of the communication between the active and regulatory sites of the two PANK3 protomers through ligand interactions with helix α10. Helix α10 of one protomer is colored orange and the other is colored teal. A, in the PANK3·acetyl-CoA complex, the ribose 3′-phosphate of acetyl-CoA interacts with the amino terminus of helix α10 in the ATP-binding site, and the CoA thioester interacts with the carboxyl terminus of helix α10 on the adjacent protomer. B, in the PANK3·AMPPNP·Mg²⁺·pantothenate complex, ATP makes strong interactions with the amino terminus of helix α10 in the nucleotide-binding site, but the substrate pantothenate does not interact with the carboxyl terminus of helix α10 on the opposite protomer resulting in a different organization of the residues in this region. C, overlay of the PANK3·AMPPNP·Mg²⁺·pantothenate (green ligands, yellow and blue structure) with the PANK3·acetyl-CoA complex (gray ligands and structure) showing the movement and side-chain rearrangements in helix α10 associated with the inactive (acetyl-CoA bound) and active (ATP bound) conformations that link the active sites of the two protomers.

FIGURE 10.

Cooperative binding of acyl-CoA to PANK3. A, inhibition of PANK3 activity by acetyl-CoA, octanoyl-CoA, and palmitoyl-CoA in the presence of 2 mm ATP. The IC₅₀ values in these experiments were as follows: acetyl-CoA, 4.6 ± 0.6 μm; octanoyl-CoA, 1.7 ± 0.08 μm, and palmitoyl-CoA, 0.5 ± 0.02 μm. B, tight binding of [³H]oleoyl-CoA to PANK3 illustrated by the co-elution of the protein and the labeled ligand on a Sepharose S-200 gel filtration column. C, cooperative binding of the fluorescent probe octanoyl-(1,N⁶)-etheno-CoA to PANK3. These data were combined from duplicate technical replicates in two independent experiments, and the average values were analyzed using the Hill equation. The lines in the graphs show the fit to the equation using the K_0.5 and Hill number parameters. D, structure of the PANK3·palmitoyl-CoA complex illustrating the intercalation of the acyl chain into the dimer interface and the altered interactions in the nucleotide binding pocket (PDB code 5KQD). E, same view of the PANK3·acetyl-CoA complex illustrate the differences with the PANK3·palmitoyl-CoA complex. The two PANK3 protomers are colored blue and yellow. F, 2F_o − F_c electron density map for the palmitoyl-CoA ligand contoured at 1σ.