Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the N-acyltransferase of lipid A biosynthesis

Abstract

LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent N-acylation of UDP-3-O-(acyl)-alpha-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 A crystal structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chlamydia trachomatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel beta-helix (LbetaH), and a C-terminal alpha-helical domain. The backbones of the LbetaH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LbetaH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LbetaH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120 degrees rotation of the C-terminal domain relative to the LbetaH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3-hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 degrees C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2'.

Figure 1. Comparison of the trimeric architecture CtLpxD (Complex I) and EcLpxD

Panels A and B show side views of the asymmetric units of CtLpxD (Complex I) (5) and EcLpxD, respectively. In both structures, chains A, B and C (green, pink and blue, respectively) of the asymmetric unit coincide with the three subunits of the physiologically relevant LpxD homotrimer. CtLpxD contains one molecule of bound free fatty acid per protein monomer (5), which is modeled as palmitate, but there is only one molecule of UDP-GlcNAc per homotrimer (carbons in yellow). The locations of the N- and C-termini are indicated for chain C. The +120° rotation of the C-terminal α-helical domain relative to the LβH domain in EcLpxD versus CtLpxD is apparent in this perspective. Panels C and D show top-down views of the asymmetric units of CtLpxD (Complex I) and EcLpxD, respectively, illustrating the precise stacking of the coils of the LβH region and the dispositions of the N-terminal nucleotide binding domains.

Figure 2. Comparison of the CtLpxD and EcLpxD monomers

Panels A and B show ribbon diagrams of chain A of CtLpxA (grey) and EcLpxD (red), respectively. The differences in the orientations of the linkers connecting the LβH and C-terminal domains of the two proteins are especially clear in this view. Panel C shows the superpositioned ribbon diagrams of the two proteins, demonstrating the close similarity of the central LβH domain, but the differences in the N-terminal and C-terminal regions. Panel D. When the N-terminal domains by themselves are superpositioned, their conformational similarities are more apparent. Aromatic side chains thought to be involved in uridine binding are indicated (4, 5).

Figure 3. Orientation of the peptide segments connecting the LβH to the C-terminal domains in CtLpxD versus EcLpxD

Panels A and B magnify and highlight the orientation of relevant chain B dipeptide for CtLpxD and the corresponding tripeptide in EcLpxD, connecting the LβH to the C-terminal region. The color scheme is the same as in Fig. 1. The end of the bound palmitate molecule present between chains A and B of CtLpxD is shown with its carbon atoms in yellow. No bound fatty acid is present in EcLpxD.

Figure 4. Proposed active sites and acyl chain binding clefts of CtLpxD versus EcLpxD

Panel A shows the superpositioned ribbons diagrams of CtLpxD (Complex I with its three protein monomers in grey) (5) and EcLpxD (chain A in green, chain B in pink, and chain C in blue). The position of the palmitate molecule between chains A and B in CtLpxA is shown in sticks, as is the catalytic base of chain A (H239 in EcLpxD and H247 in CtLpxD), the oxyanion hole (G257 in EcLpxD and G265 in CtLpxD), and the “hydrocarbon ruler” M290 of EcLpxD versus G298 of CtLpxD. The two distal carbon atoms of the palmitate chain would clash with the M290 side chain, perhaps accounting for the acyl chain selectivity of EcLpxD. Panel B shows the 2F_o—F_c map around the M290 residue in chain A of EcLpxD and the nearby peptide that connects the LβH and C-terminal regions of chain B, contoured at 1σ (see also Fig. 3B). The densities of the M290 side chain and the connecting peptide are well defined. Panel C shows the 2F_o—F_c map around the catalytic base H239 of chain A of EcLpxD, contoured at 1σ.

Figure 5. Complementation of the temperature-sensitive LpxD mutant RL25 with pWT or pM290A

Cells were streaked onto LB agar plates containing 25 μg/ml chloramphenicol and 0.1% l-arabinose and incubated overnight at 30 °C or 42 °C, as indicated. Panel A, RL25 harboring the vector control pBAD33. Panel B, RL25/pWT. Panel C, RL25/pM290A.

Figure 6. ESI/MS of lipid A from RL25 rescued at 42 °C by either pWT or pM290A

Panel A. ESI mass spectrum showing the doubly-charged [M-2H]^2- ions of the lipid A molecules from RL25/pWT grown at 42 °C. The insert shows the structure of the predominant wild-type lipid A species giving rise to the ions at m/z 897.62. Panel B. Spectrum of the doubly-charged [M-2H]^2- ions of the lipid A molecules from RL25/pM290A grown at 42 °C, showing a more complex pattern indicative of the incorporation of longer acyl chains. The peaks labeled with arrows correspond to predicted lipid A species containing either a monophosphate residue at the 1 position (blue lines) or a diphosphate group at the 1 position (red lines).

Figure 7. ESI/MS analysis of O-deacylated lipid A from RL25 rescued by pWT or pM290A

Panel A. Negative ion ESI mass spectrum of lipid A molecules from RL25/pWT grown at 42 °C. The far-right and far-left inserts show the structures of the predominant wild-type O-deacylated lipid A species, giving rise to the singly-charged ions at m/z 951.49 (blue line) and to the doubly charged ions at m/z 475.24 (black line), respectively. The peak at m/z 871.52 (green line) arises from lipid A molecules that lost their anomeric phosphate group during the mild acid hydrolysis procedure used to prepare the sample (28). Panel B. Corresponding spectrum of the O-deacylated lipid A molecules from RL25/pM290A, showing that the series of longer acyl-chains present in this material are mostly N-linked. Their masses are consistent with the incorporation of one or two R-3-hydroxypalmitate and/or R-3-hydroxy-cis-vaccenate moieties in place of R-3-hydroxymyristate at positions 2 and 2′. This series of longer chains are seen with both the singly- (blue lines) and the doubly-charged (black lines) lipid A ions, and with the 1-dephosphorylated lipid A species (green lines). The peaks noted with arrows correspond to predicted lipid A species. The remaining peaks have not been identified.

Figure 8. ESI/MS/MS analysis of O-deacylated and 1-dephosphorylated lipid A molecules obtained from RL25/pM290A

Panel A. Each of the singly-charged mono-isotopic ions indicated in green, which arise from the O-deacylated and 1-dephosphorylated lipid A molecules prepared from RL25/pM290A (Fig. 7B, green lines), were subjected to MS/MS analysis. The relevant portions of the spectra containing the ions that arise by cross-ring cleavage of the proximal unit are shown. These fragmentation ions demonstrate that the M290A variant of EcLpxD is solely responsible for the incorporation of N-linked R-3-hydroxymyristoyl, R-3-hydroxypalmitoyl, and/or R-3-hydroxy-cis-vaccenoyl chains at both the 2 and 2′ positions of lipid A, consistent with Scheme 1. Panel B. Possible structures of the fragment ions giving rise to the peaks shown above. The actual material may consist of an isobaric mixture of related cross-ring cleavage products.

Figure 9. Conservation of the LpxD hydrocarbon ruler in Gram-negative bacteria

Primary sequence alignments of various LpxD orthologs were made using Kalign (56) and are grouped together with their predicted hydroxyacyl-ACP substrates, as inferred from the dominant acyl chains attached to the 2- and 2′ positions of their lipid As (2, 10). The sequences are arranged from shortest acyl-chain length (top) to longest chain length (bottom). LpxDs that prefer fourteen-carbon or shorter hydroxyacyl-ACPs are above the line. LpxDs that prefer sixteen-carbon or longer hydroxyacyl-ACPs are below. The proposed hydrocarbon ruler region is highlighted with its methionine and leucine residues in yellow; the corresponding glycine residues of the longer chain selective LpxD variants are shown in green. Basic residues that have been proposed to interact with the hydroxyacyl-ACP donor substrate (4) are highlighted in blue. The region connecting the distal end of the LβH to the C-terminal domain (Fig. 3) is highlighted in black. An asterisk denotes the possible presence of branched-chain fatty acids.

Scheme 1

Selectivity and function of EcLpxD versus CtLpxD.

Scheme 2

Proposed catalytic mechanism of EcLpxD