Inscribing the Perimeter of the PagP Hydrocarbon Ruler by Site-Specific Chemical Alkylation

Abstract

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP selects palmitate chains using its β-barrel-interior hydrocarbon ruler and interrogates phospholipid donors by gating them laterally through an aperture known as the crenel. Lipid A palmitoylation provides antimicrobial peptide resistance and modulates inflammation signaled through the host TLR4/MD2 pathway. Gly88 substitutions can raise the PagP hydrocarbon ruler floor to correspondingly shorten the selected acyl chain. To explore the limits of hydrocarbon ruler acyl chain selectivity, we have modified the single Gly88Cys sulfhydryl group with linear alkyl units and identified C10 as the shortest acyl chain to be efficiently utilized. Gly88Cys-S-ethyl, S-npropyl, and S-n-butyl PagP were all highly specific for C12, C11, and C10 acyl chains, respectively, and longer aliphatic or aminoalkyl substitutions could not extend acyl chain selectivity any further. The donor chain length limit of C10 coincides with the phosphatidylcholine transition from displaying bilayer to micellar properties in water, but the detergent inhibitor lauryldimethylamine N-oxide also gradually became ineffective in a micellar assay as the selected acyl chains were shortened to C10. The Gly88Cys-S-ethyl and norleucine substitutions exhibited superior C12 acyl chain specificity compared to that of Gly88Met PagP, thus revealing detection by the hydrocarbon ruler of the Met side chain tolerance for terminal methyl group gauche conformers. Although norleucine substitution was benign, selenomethionine substitution at Met72 was highly destabilizing to PagP. Within the hydrophobic and van der Waals-contacted environment of the PagP hydrocarbon ruler, side chain flexibility, combined with localized thioetheraromatic dispersion attraction, likely influences the specificity of acyl chain selection.PagP is an outermembrane palmitoyltransferase that catalyzes the transfer of a palmitate chain (16:0) fromthe sn-1 position of a glycerophospholipid to the free hydroxyl group of the (R)-3-hydroxymyristate chain at position 2 of lipid A (endotoxin) ( Figure 1A) CIHR Author Manuscript CIHR Author Manuscript CIHR Author ManuscriptEscherichia coli and related pathogenic Gram-negative bacteria, PagP evades host immune defenses by resisting antimicrobial peptides (4-7) and attenuating the inflammatory response to infection triggered by lipopolysaccharide through the TLR4/MD2 pathway (8-10). Transcription of the pagP gene is governed by the PhoP/PhoQ regulon, which controls expression of bacterial lipid A modification enzymes induced during infection by host antimicrobial peptides (11,12). PagP is thus a target for the development of anti-infective agents and a tool for the synthesis of novel vaccine adjuvants and endotoxin antagonists (2, 3).The structure of E. coli PagP has been determined both by solution nuclear magnetic resonance spectroscopy and by X-ray crystallography, revealing an eight-strand antiparallel β-barrel preceded by an N-terminal amphipathic α-helix (13-...

A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis‐specific lipid A

“…Hydrophobic residues on the exterior of the protein are located where they would be embedded in the membrane, while the hydrophilic portions are at the expected solvent‐exposed membrane interface regions. The terminal six methylene units of the palmitate chain maintain van der Waals contacts with the E. coli hydrocarbon ruler (Khan et al ., 2010b) and a similar interaction is apparent in PA PagP (Fig. A and B).…”

“…B). Since detergents that effectively mimic the structures of fatty acyl chains are E. coli PagP competitive inhibitors, only detergents with bulky substituents that sterically preclude their binding within the hydrocarbon ruler are able to support the lipid A palmitoyltransferase reaction (Khan et al ., 2010a,b). As shown in Fig.…”

“…C). The results demonstrate that PA PagP clearly shares E. coli PagP's unique ability to discriminate acyl chains that differ from palmitate by only a single methylene unit (Khan et al ., 2010a,b). Based on the PA PagP homology model, His35, like His33 in E. coli PagP, maps to the cell surface region adjacent to the hydrocarbon ruler.…”

UVliPiD: A UVPD-Based Hierarchical Approach for De Novo Characterization of Lipid A Structures

“…40,41 Interestingly, the relative abundance of the acyl loss fragment ions suggests that incorporation of the 13:0 fatty acid chain at the 2′ position was the most favorable. 40,41 Identification and characterization of these isomeric structures highlights the utility of a rapid, database-independent lipid A analysis strategy.…”

Kdo2‐lipid A: structural diversity and impact on immunopharmacology

“…PagP has been well characterized in both E. coli and Salmonella Typhimurium, and its structure has been determined by both NMR spectroscopy and X-ray crystallography (Hwang, Bishop & Kay, 2004; Bishop, 2008). Palmitate is selected specifically by PagP, employing a gating mechanism sensitive to the length of hydrocarbon chains of potential donor lipids (Khan et al , 2010). …”