ABSTRACT
The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP selects palmitate chains using its ß-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-n-propyl, 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 thioether-aromatic dispersion attraction, likely influences the specificity of acyl chain selection.
Subject(s)
Acyltransferases/chemistry , Bacterial Outer Membrane Proteins/chemistry , Escherichia coli Proteins/chemistry , Acyltransferases/genetics , Acyltransferases/metabolism , Alkylation , Amino Acid Substitution , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Base Sequence , Binding Sites/genetics , Circular Dichroism , DNA Primers/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Lipid A/chemistry , Lipid A/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
We report here the first study of the protonated, neutral form of tetrazoles as anion binding functional groups. Our studies reveal them to be capable of binding anions with extremely high potency in polar solutions. In studying carboxylic acid-containing congeners, we find a remarkable discrepancy: a strictly analogous acid-containing host binds anions > or = 50,000-fold more weakly than the tetrazole under study. We can explain this functional difference by considering tetrazole tautomerization equilibria and carboxylic acid conformational preferences.
Subject(s)
Carboxylic Acids/chemistry , Molecular Conformation , Tetrazoles/chemistry , Anions/chemistry , Magnetic Resonance Spectroscopy , Models, MolecularABSTRACT
A new synthetic model of arginine-carboxylate-aromatic triads-common motifs at sites of protein-protein interactions-is reported. Binding studies in mixed methanol/water solvent systems suggest that the carboxylate-binding ability of pi-stacked guanidinium ions is improved relative to a non-stacked control.
