Substrate-induced asymmetry and channel closure revealed by the apoenzyme structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase

Abstract

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in prokaryotic coenzyme A (CoA) biosynthesis, directing the transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to yield dephospho-CoA (dPCoA). The crystal structures of Escherichia coli PPAT bound to its substrates, product, and inhibitor revealed an allosteric hexameric enzyme with half-of-sites reactivity, and established an in-line displacement catalytic mechanism. To provide insight into the mechanism of ligand binding we solved the apoenzyme (Apo) crystal structure of PPAT from Mycobacterium tuberculosis. In its Apo form, PPAT is a symmetric hexamer with an open solvent channel. However, ligand binding provokes asymmetry and alters the structure of the solvent channel, so that ligand binding becomes restricted to one trimer.

Figure 1.

The crystal structure of M. tuberculosis phosphopantetheine adenylyltransferase (PPAT). (A) Stereocartoon drawing of the structure of the tuberculin PPAT protomer. αHelices are depicted by light blue helical ribbons and β strands by yellow arrows. The N terminus of each helix is marked with a positive sign while the C terminus of each helix is indicated with a negative sign, in agreement with the helix dipole moment. As in the case of other dinucleotide binding folds, PPAT is folded into two symmetrically related halves. The first half consists of three βstrands (β1, β2, and β3) connected by intervening β-helices (α1 and α2), while the second half contains two β strands (β4 and β5) related by α-helices (α3 and α4). A tandem repeated motif consisting of a short 3₁₀-helix followed by an α-helix (α5 and α6) comprises the C terminus. A short linker sequence connects the two halves of the fold. The catalytic histidine is shown in ball-and-stick representation. (B) Stereo Cα-trace superposition of tuberculin (black) PPAT onto coliform PPAT bound to ligands (red; Ppant or dPCoA bound subunits) and unliganded coliform PPAT (green). The view shown is the same view as that presented in (A). The largest discrepancies between the apo-form of PPAT (black) and the liganded subunits of E. coli PPAT (red) is found for residues located on the loop following β-strands β2 and β4. The largest difference between the M. tuberculosis apo-form PPAT structure and the E. coli PPAT structures is found for the loop connecting strand β2 with helix α2, which closes over the active site.

Figure 2.

Stereo superposition of the E. coli PPAT structure bound to dPCoA onto the M. tuberculosis PPAT apo-structure. Residues involved in binding to dPCoA or pyrophosphate, as seen in the E. coli PPAT:dPCoA structure, are shown in red and the equivalent residues found in the M. tuberculosis are shown in blue. The product (dPCoA) is shown in ball-and-stick representation. Movements of the catalytic lysine are indicated.

Figure 3.

Cα trace superposition of the of E. coli PPAT (three shades of gray) hexamer as seen in the PPAT:dPCoA structure onto the apo-form of M. tuberculosis PPAT (three shades of red). The N and C termini are indicated. For clarity, the hexamer is depicted in two halves of trimers. (A) The 195 Cα positions of residues forming β strands or α helices of the unliganded E. coli PPAT trimer can be superimposed onto the equivalent Cα positions of the apo-from of M. tuberculosis PPAT with RMSD of 0.9 Å.

Figure 4.

Stereo electrostatic surface potential of the PPAT hexamer along the triad looking into the solvent channel. (A) The opening of the M. tuberculosis symmetric hexamer in its apo-form is much wider than that seen for the (B) unliganded E. coli PPAT trimer or the (C) liganded trimer within the asymmetric E. coli PPAT bound hexamer. The view of (C) is from the bottom of the view shown in (B); in other words, the view in (C) is rotated by 180° around a horizontal axis (red, negative; blue, positive; white, uncharged).