The structure of Staphylococcus aureus phosphopantetheine adenylyltransferase in complex with 3'-phosphoadenosine 5'-phosphosulfate reveals a new ligand-binding mode

Abstract

Bacterial phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway. It catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate. Previous structural studies have revealed how several ligands are recognized by bacterial PPATs. ATP, ADP, Ppant and dPCoA bind to the same binding site in a highly similar manner, while CoA binds to a partially overlapping site in a different mode. To provide further structural insights into ligand binding, the crystal structure of Staphylococcus aureus PPAT was solved in a binary complex with 3'-phosphoadenosine 5'-phosphosulfate (PAPS). This study unexpectedly revealed a new mode of ligand binding to PPAT, thus providing potentially useful information for structure-based discovery of inhibitors of bacterial PPATs.

Figure 1

Stereo ribbon diagram of S. aureus PPAT monomer. PAPS bound in the active site is shown as a stick model.

Figure 2

F _o − F _c OMIT electron-density map of PAPS contoured at the 2.5σ level. The mean B factor of PAPS (40.8 Ų) is slightly higher than that of the protein atoms, suggesting that the occupancy of PAPS is slightly lower than one. When the occupancy of PAPS is fixed at 0.8, its B factor (30.9 Ų) is comparable to that of the protein atoms (28.6 Ų).

Figure 3

Stereoview of the active site around the bound PAPS.

Figure 4

Alignment of bacterial PPAT amino-acid sequences. SA, Staphylococcus aureus (SWISS-PROT accession code P63819); EF, Enterococcus faecalis (Q831P9); TM, Thermotoga maritima (Q9WZK0); EC, Escherichia coli (P0A6I6); MT, Mycobacterium tuberculosis (P0A530); TT, Thermus thermophilus (Q72K87). Strictly conserved residues and semi-conserved residues are coloured cyan and yellow, respectively. The TXGH sequence motif is enclosed in a red box. The residues of S. aureus PPAT that interact with PAPS are indicated by blue inverted triangles above the sequences. Cylinders and arrows above the sequences denote α-helices and β-strands, respectively. Secondary-structure elements are indicated for the PAPS complex of S. aureus PPAT. This figure was produced using ClustalX (Thompson et al., 1997 ▶) and GeneDoc (http://www.nrbsc.org/).

Figure 5

The distinct mode of PAPS (blue) binding to S. aureus PPAT. ATP (yellow, 1gn8 chain B) and dPCoA (orange, 1b6t chain B) bound in E. coli PPAT (Izard & Geerlof, 1999; Izard, 2002 ▶) are positioned in the active site of S. aureus PPAT by superimposing PPAT monomer structures. With the exception of Fig. 4 ▶, all figures were produced using PyMOL (http://www.pymol.org).