Abstract
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 β-strand-to-α-helix and α-helix-to-β-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.
Highlights
Phosphatidyl-myo-inositol mannosyltransferase A (PimA), is an essential GT-B enzyme involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs),3 lipomannan, and lipoarabinomannan, key structural elements and virulence factors of Mycobacterium tuberculosis [1,2,3,4,5,6]
This result clearly indicates that PimA aromatic side chains gain freedom in the presence of diC8PI micelles
To investigate the impact of phospholipids binding into the conformation of PimA, we monitored the tertiary structure changes of PimA induced by the addition of SUVs made of DOPC, DOPG, and PI, by near-UV CD (Fig. 2A)
Summary
Phosphatidyl-myo-inositol mannosyltransferase A (PimA), is an essential GT-B enzyme involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs),3 lipomannan, and lipoarabinomannan, key structural elements and virulence factors of Mycobacterium tuberculosis [1,2,3,4,5,6]. Inspection of the crystal structure of PimA suggested that the presence of an amphipathic ␣-helix (␣2-helix; residues 72– 87) and surface-exposed hydrophobic residues in the N-terminal domain of the protein could mediate the association of PimA with anionic phospholipids via electrostatic interactions [11].
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