RESUMEN
The cytoplasmic membrane is probably the most important physical barrier between microbes and the surrounding habitat. Aminoacylation of the polar head group of the phospholipid phosphatidylglycerol (PG) catalyzed by Ala-tRNA(Ala)-dependent alanyl-phosphatidylglycerol synthase (A-PGS) or by Lys-tRNA(Lys)-dependent lysyl-phosphatidylglycerol synthase (L-PGS) enables bacteria to cope with cationic peptides that are harmful to the integrity of the cell membrane. Accordingly, these synthases also have been designated as multiple peptide resistance factors (MprF). They consist of a separable C-terminal catalytic domain and an N-terminal transmembrane flippase domain. Here we present the X-ray crystallographic structure of the catalytic domain of A-PGS from the opportunistic human pathogen Pseudomonas aeruginosa. In parallel, the structure of the related lysyl-phosphatidylglycerol-specific L-PGS domain from Bacillus licheniformis in complex with the substrate analog L-lysine amide is presented. Both proteins reveal a continuous tunnel that allows the hydrophobic lipid substrate PG and the polar aminoacyl-tRNA substrate to access the catalytic site from opposite directions. Substrate recognition of A-PGS versus L-PGS was investigated using misacylated tRNA variants. The structural work presented here in combination with biochemical experiments using artificial tRNA or artificial lipid substrates reveals the tRNA acceptor stem, the aminoacyl moiety, and the polar head group of PG as the main determinants for substrate recognition. A mutagenesis approach yielded the complementary amino acid determinants of tRNA interaction. These results have broad implications for the design of L-PGS and A-PGS inhibitors that could render microbial pathogens more susceptible to antimicrobial compounds.
Asunto(s)
Aminoaciltransferasas/química , Bacillus/enzimología , Proteínas Bacterianas/química , Fosfatidilgliceroles/metabolismo , Pseudomonas aeruginosa/enzimología , Factores R , ARN de Transferencia de Alanina/metabolismo , ARN de Transferencia de Lisina/metabolismo , Aminoacilación , Aminoaciltransferasas/metabolismo , Bacillus/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Lisina/biosíntesis , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Fosfatidilgliceroles/biosíntesis , Conformación Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusión/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
An evanescent wave fiber optic sensor for detection of Escherichia coli (E. coli) outer membranes proteins (EcOMPs) using long period gratings (LPGs) as a refractometric platform is presented. The sensing probes were attained by the functionalization of LPGs inscribed in single mode fiber using two different methods of immobilization; electrostatic assembly and covalent binding. The resulting label-free configuration enabled the specific recognition of EcOMPs in water by monitoring the resonance wavelength shift due to refractive index changes induced by binding events. The sensors displayed linear responses in the range of 0.1 nM to 10 nM EcOMPs with sensitivities of -0.1563±0.005 nm decade(-1) [EcOMP, M] (electrostatic method) and -0.1597±0.004 nm decade(-1) [EcOMP, M] (covalent method). The devices could be regenerated (under low pH conditions) with a deviation less than 0.1% for at least three subsequent detection events. The sensors were also applied to spiked environmental water samples.