Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Abstract

UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 A in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel beta-helix fold. We now present the crystal structure at 1.8 A resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a beta-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki = 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact beta-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.

Fig. 1.

Function of LpxA in lipid A biosynthesis. LpxA catalyzes the first step of lipid A biosynthesis. It transfers the R-3-hydroxyacyl moiety from R-3-hydroxyacyl-ACP to the 3-position of UDP-GlcNAc. E. coli LpxA is highly selective for 14 carbons (8). The glucosamine ring is colored blue, and the 3-position is indicated. Kdo₂-lipid A is the minimal lipopolysaccharide that supports growth in most bacteria (9). The acyl chains at the 3 and 3′ positions of Kdo₂-lipid A are derived from LpxA.

Fig. 2.

Crystal structure of the LpxA–peptide 920 complex at 1.8 Å. (A) In this side view, the individual LpxA subunits of the homotrimer are colored pink, green, and blue. The LpxA N termini, located at the bottom, form the start of the β-helix domain of each subunit. Peptide 920 (yellow) is in a β-hairpin conformation with its N and C termini protruding. In the space-filling model, peptide 920 carbons are yellow, nitrogens blue, and oxygens red. (B) Top-down view of LpxA with bound peptide 920. The threefold symmetry and precise stacking of the 18-aa residues in each β-helix coil are visualized clearly. Arrows point to protruding peptide 920. (C) Side view of a space-filling model of LpxA, highlighting the relative positions of H125 and peptide 920, which are not in direct contact. (D) Top-down view of free LpxA, docked computationally to butyryl-ACP based on NMR constraints (35). The positions of H125, R204 (a proposed contact site between LpxA and ACP), and S36 of ACP are indicated. The location of the butyryl phosphopantetheine moiety, which is attached to S36, was not included in the published model (16).

Fig. 3.

Inhibition of LpxA activity by peptide 920. Peptide 920 at the indicated concentrations was preincubated in the assay mixture at 30°C for 3 min. The reaction was initiated by the addition of 1 nM LpxA. The data were fit to Eqs. 1 or 2 for IC₅₀ determination. (A) The ≈12-fold shift in the IC₅₀ indicates that peptide 920 is competing with acyl-ACP. The green squares show that the IC₅₀ is 60 ± 9 nM when acyl-ACP is 1 μM. The blue circles show a shift in IC₅₀ to 730 ± 107 nM when acyl-ACP is 100 μM. In both cases, UDP-GlcNAc was 1 μM. The initial rate that corresponds to 100% at 1 μM acyl-ACP is 2.8 ± 0.4 nmol·min⁻¹mg⁻¹. At 100 μM acyl-ACP, it is 6.6 ± 1.3 nmol·min⁻¹mg⁻¹. (B) Peptide 920 does not compete effectively with UDP-GlcNAc, because increasing UDP-GlcNAc from 1 μM (green) to 500 μM (blue), or even to 5 mM (red), does not change the IC₅₀ significantly with acyl-ACP held at 50 μM. The initial rate that corresponds to 100% at 1 μM UDP-GlcNAc is 7.1 ± 0.8 nmol·min⁻¹mg⁻¹. At 500 μM UDP-GlcNAc, it is 4.4 × 10² ± 1.4 × 10² nmol·min⁻¹mg⁻¹. At 5 mM UDP-GlcNAc, it is 2.3 × 10³ ± 4.9 × 10² nmol·min⁻¹mg⁻¹. Error bars show SDs of triplicates.

Fig. 4.

Noncovalent interactions between peptide 920 and LpxA. (A) Stereoview of the final 2 F_o − F_c electron density map contoured at 1 σ around peptide 920. The peptide is shown as a stick model with carbons in yellow. (B) Stereoview of important protein–peptide 920 interactions. The peptide 920 carbons are colored yellow, whereas the LpxA subunit carbons are either green or pink, according to the coloring scheme in Fig. 2. All of the N and O atoms are blue and red, respectively. Hydrogen bonds are green dashed lines. Water molecules are cyan. Hydrogen bonds involving water are not included for clarity. Only one of two possible conformations is shown for the R258 and M170 side chains. The peptide is rotated ≈90° clockwise along its vertical axis when compared with A. (C) Schematic drawing of key interactions between peptide 920 and LpxA. Hydrogen bonds are shown as green dashes. A van der Waals interaction is indicated by the black arrowhead. Water molecules are cyan balls.

Fig. 5.

Location of peptide 920 and conserved residues implicated in catalysis. (A) View into one of the three peptide-binding cavities of the LpxA trimer in the absence of peptide 920. LpxA subunits are colored pink, green, or blue (not visible) as in Fig. 2. Key residues implicated in catalysis or substrate binding by mutagenesis are colored according to element with carbons in gray. (B) Same view as above but with peptide 920 present in yellow. Conserved LpxA residues are labeled in white. There is some space between peptide 920 and the conserved residues H125, H144, H122, and K76. Because of the hydrogen bonding of peptide 920 with G173 and H160, these residues are largely hidden from view. In the complete model, a few water molecules are located in the space beneath peptide 920 (data not shown).