Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis

Abstract

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein's invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.

Figure 1. Lipoprotein posttranslational processing in Gram-negative bacteria.

The pathway includes the three processing enzymes Lgt, LspA and Lnt that reside in the inner membrane. Pre-prolipoproteins enter the membrane for processing via the Sec or TAT pathways. Trafficking to the outer membrane is by way of the Lol ABC transporter system. The inset shows the chemical structure of the triacylated N-terminal cysteine of a mature lipoprotein. DAG, diacylglyceryl.

Figure 2. Lnt activity assay.

(a–c) Lnt activity monitored as a shift in the SDS-PAGE mobility of FSL-1-biotin on N-acylation with quantitation by western blotting. Uncropped images of the western blots including marker lanes are shown in Supplementary Fig. 1. (d,e) Lnt activity monitored by the conversion of NBD-PE to NBD-lyso-PE (NBD-LPE) by thin layer chromatography with quantitation by fluorescence. (a) N-acyl transferase activity of LntPae and LntEco is evident as a band shift towards higher molecular weight values resulting from a conversion of FSL-1-biotin (open arrow head) to N-acylated FSL-1-biotin (full arrow head) in the presence of a lipid donor. (b) Time course experiment and lipid head group specificity of LntPae. After a 1 h incubation at 37 °C in the presence of both substrates and enzyme (20 nM), the FSL-1-biotin band appeared ∼3 mm higher in the gel than was observed for both negative control reactions without either DOPE or LntPae (lanes 1 and 2). The time dependence of the reaction is shown in lanes 3–5. When DOPE was replaced with DOPG or DOPC, the product was formed much less efficiently (lanes 6 and 7). (c) Densitometric analysis of the time-dependent data in b. (d) LntEco activity measurements for wild type (WT) and mutants E267Q, K335A and C387S. The reaction was stopped after 60 min. Data are shown for duplicate reaction measurements. (e) Time course of lyso-PE production catalysed by LntEco. (f) Densitometric analysis of the time-dependent data in e.

Figure 3. Overall architecture of Lnt from E. coli.

(a) View from the membrane plane. The protein has two domains, a membrane domain and a periplasmic nitrilase-like domain. The structure is shown in cartoon representation and rainbow colour coded from N (blue) to C terminus (red). The catalytic cysteine Cys387 side chain is shown in sphere representation (carbon, magenta; sulfur, yellow). The magenta arrow indicates the proposed substrate entry portal and identifies what is referred to as the front of the enzyme. Approximate location of the membrane boundaries are shown as horizontal lines. Cys387 sits ∼13 Å above the bulk membrane surface. (b) Schematic representation of the secondary structure elements in the LntEco structure. Colour coding follows that used in a.

Figure 4. Membrane domain of LntEco.

(a) View from the membrane plane. (b) View from the periplasm. (c) View from the cytoplasm. Colour coding as in Fig. 3. Gly74 and Pro129 side chains shown as spheres.

Figure 5. Nucleophilic elbow in LntEco.

(a) The nucleophilic elbow (orange) consisting of a β-strand-turn-helix (dashed box) shown in context of the overall Lnt structure (grey). (b) Expanded view of the boxed region in a showing the catalytic Cys387 in the turn. The oxyanion hole created by backbone amides in α3′ is indicated. (c) Coordination between residues in the turn and the α3′ helix. Dashed lines correspond to distances of ≤3.5 Å. (d) Residues in L1 are coordinated to the core of the MD via Arg139 in H5 and to the nucleophilic elbow via Tyr388.

Figure 6. Nitrilase-like domain of LntEco.

(a) View from the membrane plane as in Fig. 3a. Colour coded by secondary structure to highlight the αββα sandwich feature of the domain. Catalytic triad residues are shown in stick representation. The asterisks in α** and β5** indicate that the α-helix and β-strand secondary structure elements form in some structures (α-helix found in: LntEco C387A, chain A; LntPae WT. β-strand found in: LntPae WT) but not in others (α-helix absent in: LntEco WT, chains A and B; LntEco C387A, chain B). (b) Schematic representation of the secondary structure elements in the nitrilase-like domain. Colour coding follows that used in a. Helix α3 consists of two small helices, α3′ and α3″. The dashed lines around α** and β5 indicate that these elements are formed in some structures but not in others as in a.

Figure 7. Architecture of LntEco active site pocket.

(a) View into the active site pocket from the membrane surface. Helices and loops, referred to as arms, extending from the MD and the ND radiate out creating a funnel-shaped entrance to the active site. Spheres are used to mark the reach of each arm. Reaches are connected to the catalytic Cys387 by dashed lines to communicate a sense of the funnelled nature of the entrance. A description of what constitutes the different arms follows. Arm 1. Periplasmic extensions of H3 and H4 and connecting loop, originating in the MD. Arm 2. L1 between H5 and H6 in the MD. Arm 3. The 40-residue long loop between β5 and β6 linking the two halves of the αββα sandwich in the ND. Arm 4. Long β-strands β3 and β4 and connecting loop in the upper half of the ND sandwich. Arm 5. Loop connecting β1 and α2 in upper half of ND sandwich. Arm 6. Loop between β9 and β10 in bottom half of ND sandwich. Arm 7. Loop between β1 and α1 in upper half of ND sandwich. Arm 8. Loop between β11 and β12, connecting the bottom and top halves of the sandwich. Arm 9. Loop between β8 and α4 in bottom half of the ND sandwich. (b) Expanded view into the active site showing the catalytic triad residues, E267, K335 and C387 along with other proximal conserved residues. Side chains are shown in stick representation. The orientation is similar to that in a. Dashed lines correspond to distances of ⩽3.2 Å.

Figure 8. Bound lipids define substrate portal in and membrane around Lnt.

(a) Structured monoolein lipids in LntEco-C387A decorate the surface of the protein in a manner reminiscent of the membrane bilayer and the portal between the bulk membrane and the enzyme active site. Lipid molecules in the portal line up in single file and are individually numbered from 1 to 4. Lipids are shown in stick representation. Cys387Ala is coloured magenta. (b) Expanded view of lipids arranged in single file in the portal facing into the active site and of lipids (shown in stick representation) at the surface of the enzyme. (c) Lipid binding to the surface and in the portal of the enzyme. Enzyme shown in surface representation with hydrophobic residues in light blue and polar residues in grey. Orthogonal views presented in left, middle and right panels. An expanded view of lipids (shown as spheres) in the portal is shown in the right panel.

Figure 9. Proposed N-acyltransferase reaction mechanism in Lnt.

Electron lone pairs shown as double dots. (a) First Michaelis complex with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) substrate. (b) First tetrahedral intermediate. Tetrahedral carbon shown in stereochemical representation. The oxyanion bears a negative charge. (c) Second Michaelis complex with apo-lipoprotein substrate. (d) Second tetrahedral intermediate. (e) Product complex. (f) Empty active site ready to undergo another reaction cycle. Red curved arrows indicate electron flow. Dashed blue lines denote oxyanion stabilization. GPE, glyceryl-phosphoethanolamine; LP, apo-lipoprotein; DAG, diacylglyceryl; LPE, lyso-PE.