RESUMEN
Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes ß-strand-to-α-helix and α-helix-to-ß-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.
Asunto(s)
Proteínas Bacterianas/metabolismo , Manosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Mycobacterium smegmatis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dicroismo Circular , Escherichia coli/genética , Manosiltransferasas/química , Manosiltransferasas/genética , Proteínas de la Membrana/química , Modelos Moleculares , Fosfolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including ß-strand-to-α-helix and α-helix-to-ß-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.
Asunto(s)
Proteínas Bacterianas/química , Membrana Celular/metabolismo , Glicosiltransferasas/metabolismo , Manosiltransferasas/química , Estructura Secundaria de Proteína , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Membrana Celular/enzimología , Cristalografía por Rayos X , Humanos , Manosiltransferasas/genética , Manosiltransferasas/aislamiento & purificación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfolípidos/metabolismo , Estructura Secundaria de Proteína/genéticaRESUMEN
The FapR protein of Bacillus subtilis has been shown to play an important role in membrane lipid homeostasis. FapR acts as a repressor of many genes involved in fatty acid and phospholipid metabolism (the fap regulon). FapR binding to DNA is antagonized by malonyl-CoA, and thus FapR acts as a sensor of the status of fatty acid biosynthesis. However, malonyl-CoA is utilized for fatty acid synthesis only following its conversion to malonyl-ACP, which plays a central role in the initiation and elongation cycles carried out by the type II fatty acid synthase. Using in vitro transcription studies and isothermal titration calorimetry, we show here that malonyl-ACP binds FapR, disrupting the repressor-operator complex with an affinity similar to that of its precursor malonyl-CoA. NMR experiments reveal that there is no protein-protein recognition between ACP and FapR. These findings are consistent with the crystal structure of malonyl-ACP, which shows that the malonyl-phosphopantetheine moiety protrudes away from the protein core and thus can act as an effector ligand. Therefore, FapR regulates the expression of the fap regulon in response to the composition of the malonyl-phosphopantetheine pool. This mechanism ensures that fatty acid biosynthesis in B. subtilis is finely regulated at the transcriptional level by sensing the concentrations of the two first intermediates (malonyl-CoA and malonyl-ACP) in order to balance the production of membrane phospholipids.
Asunto(s)
Proteína Transportadora de Acilo/química , Ácidos Grasos/biosíntesis , Proteína Transportadora de Acilo/genética , Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Malonil Coenzima A/química , Modelos Moleculares , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid biosynthesis. The molecular mechanisms of regulation of the acpP structural gene, as well as the function of its gene product, are poorly characterized in Bacillus subtilis and other Gram-positive organisms. Here, we report that transcription of acpP takes place from two different promoters: PfapR and PacpP. Expression of acpP from PfapR is coordinated with a cluster of genes involved in lipid synthesis (the fapR operon); the operon consists of fapR-plsX-fabD-fabG-acpP. PacpP is located immediately upstream of the acpP coding sequence, and is necessary and sufficient for normal fatty acid synthesis. We also report that acpP is essential for growth and differentiation, and that ACP localizes in the mother-cell compartment of the sporangium during spore formation. These results provide the first detailed characterization of the expression of the ACP-encoding gene in a Gram-positive bacterium, and highlight the importance of this protein in B. subtilis physiology.