A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin

Abstract

The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl-chain recognition in any membrane-bound enzyme remains to be defined. PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin). PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler. We present the 1.9 A crystal structure of PagP, an eight-stranded beta-barrel with an unexpected interior hydrophobic pocket that is occupied by a single detergent molecule. The buried detergent is oriented normal to the presumed plane of the membrane, whereas the PagP beta-barrel axis is tilted by approximately 25 degrees. Acyl group specificity is modulated by mutation of Gly88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between PagP and the lipocalins suggests an evolutionary link between these proteins.

Figure 1

The asymmetric outer membrane of Gram-negative bacteria consists of an inner phospholipid leaflet (indicated by the two molecules of phosphatidylethanolamine in the lower part of the panel) and an outward-facing leaflet of lipid A bearing minimally two 3-deoxy-D-manno-2-octulosonic acid (Kdo) sugars (indicated by a Kdo₂-lipid A in the upper part of the panel, shown as the palmitoylated product of the PagP reaction). PagP catalyzes the transfer of a palmitate chain (shown in red) from the sn-1 position of a phospholipid to the lipid A acceptor. This transfer requires the migration of the phospholipid donor to the outer leaflet of the outer membrane, presumably by mechanisms that do not directly involve PagP.

Figure 2

Ribbon diagrams of (A) PagP, (B) OmpA (Protein Data Bank code 1QJP; Pautsch and Schulz, 2000), and (C) NspA (Protein Data Bank code 1P4T; Vandeputte-Rutten et al, 2003).

Figure 3

(A) Superposition of the PagP crystal structure (red) with the 20 lowest energy NMR structures in DPC micelles (black) (Hwang et al, 2002; Protein Data Bank code 1MM4). The leading α-helix is at the lower right, and loop L1 is at the upper left. (B) Ribbon diagram of PagP. 2∣∣F_o∣−∣F_c∣∣ electron density for the internal LDAO ligand is shown in gray mesh, and the aromatic residues located at the presumed membrane/water interfaces are shown in black. Residues 38–47 of extracellular loop L1 are not visible in the electron density maps and are indicated by the dashed segment. The barrel axis is tilted approximately 25° from the membrane normal and is shown as a red line. The horizontal lines represent the presumed position of the outer membrane bilayer. (C) Hydrophobicity profiles (Wimley, 2002) for the outward-facing PagP residues as a function of membrane position are shown as solid lines. Negative Σ(ΔG) values indicate regions that are more hydrophobic. The gray line and symbols present results for the protein positioned with the β-barrel axis aligned along the membrane normal, while the black line and symbols are for the protein tilted as in panel B. The solid symbols represent the Cγ positions of the Trp (squares), Tyr (circles), and Phe (triangles) residues that form the inner (Trp17, Tyr23, Trp89, Trp93, Phe101, Tyr133, Phe161) and outer (Trp32, Trp51, Trp81, Tyr119, Tyr142, Tyr153) aromatic belts. (D) The hydrophobicity profiles for the inward-facing residues of PagP (black), OmpA (blue), and NspA (red) indicate an unusually hydrophobic interior in the upper half of the PagP β-barrel.

Figure 4

(A) Multiple sequence alignment of PagP homologues from E. coli, Salmonella typhimurium, Erwinia chrysanthemi, Yersinia pestis, Photorhabdus luminescens, Legionella pneumophila, and Bordetella pertussis. To date, the enzyme has been found primarily in pathogenic Gram-negative bacteria, and a representative species from each genus is included in the alignment. Residues highlighted in blue boxes are identical in all known homologues, and conserved residues are shown in yellow boxes. The putative catalytic residues are included in black boxes. Residues that are located within the bilayer are indicated with horizontal green bars, with the lipid-exposed positions shown in darker green. The residue numbering is based on the E. coli PagP sequence of the mature protein without the leading signal sequence. Disordered regions in the crystal structure are indicated by the dashed lines. The N-terminal amino acid of the mature E. coli protein was determined by Edman degradation (Bishop et al, 2000). The N-terminal amino acids of the other homologues following the removal of the signal sequences have not yet been experimentally determined. (B) An ‘unrolled' barrel was generated by projecting atomic positions onto a cylinder aligned with the barrel axis and laid flat. The light green shading indicates the position of the membrane, which follows a sinusoidal path because of the tilting of the barrel in the bilayer. One central PagP molecule is drawn with green carbon atoms, while flanking strands are shown with white carbon atoms. Residues 8–20 of the leading α-helix and residues 33–37 and 48–50 from L1 are omitted for clarity. Side chains are omitted, except for those of prolines 28 and 127 and residues from the aromatic belts. Interchain hydrogen bonds are drawn with dashed lines.

Figure 5

Ligand interactions in PagP. (A) Cut-away view of the protein, showing the inner ligand pocket and the internally bound detergent molecule. (B) Overhead view of the empty barrel in surface representation, colored according to the electrostatic surface potential. (C) Local environment of the internal LDAO detergent. Side-chain Cα carbons are indicated in black. (D) Strands F and G are shown in stick representation, and form main-chain interstrand H bonds only in the bottom part of the barrel. In the upper part of the barrel, the space between the two strands is filled with side chains from apolar residues. Hydrogen bonds are indicated with dashed lines, and the side chains for residues Trp117, Leu122, Val124, Leu126, Pro127 (strand F) and Thr141, Pro144, and Tyr147 (strand G) are shown in black.

Figure 6

Enzymatic activity of PagP. (A) LDAO and DPC are relatively compact amphiphiles, while CYFOS-7 and DDM have bulky tail and head groups, respectively. (B) PagP is active in CYFOS-7 and DDM, but shows little activity in DPC or LDAO. (C) PagP in 0.25% DDM is inhibited by DPC and LDAO. (D) The PagP donor acyl-chain specificity in phosphatidylcholine (PtdCho) is a function of the amino-acid side chain at position 88. The wild-type protein has Gly88 and preferentially transfers a palmitate group to Kdo₂-lipid A. The G88A, G88C, and G88M mutants provide side chains that occupy the acyl-binding pocket of PagP to progressively greater extents, and decrease the specificity of the transferred chain by 1, 2, and 4 methylene equivalents, respectively.

Figure 7

Superposition of the PagP (blue) and retinol-binding protein (red, Protein Data Bank code 1AQB) structures. The LDAO (blue) and retinol (red) ligands are shown in ball-and-stick representation. PagP has an N-terminal α-helix, while most lipocalin structures, including RBP, have a single α-helix at the C-terminus.