Title
Molecular basis of membrane association by the phosphatidylinositol mannosyltransferase PimA from mycobacteria
Author
Ane Rodrigo-Unzueta, Mariano A. Martínez, Natalia Comino, Pedro M. Alzari, Alexandre Chenal, Marcelo E. Guerin
Year
2016
Journal
The Journal of Biological Chemistry
Abstract
Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM), which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated GTs 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 (PI) substrate and non-subtrate membrane model systems (SUVs) 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 amphipatic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid head groups 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 amphipatic 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 towards the dynamic and highly fluctuating lipid bilayer.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Tertiary structure, Vesicle interactions, Thermal stability, Ligand binding, Biochemistry