Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria
Journal of Biological Chemistry
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 peripheral 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 used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple-step mechanical unfolding pathways at low force, akin to molten globule states. Interestingly, the ab initio lowresolution envelopes obtained from small-angle Xray scattering of the unliganded PimA and the PimA-GDP complexed forms clearly demonstrate that not only the ′open′ and ′closed′ conformations of the GT-B enzyme are largely present in solution but, in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal ′Rossmann fold′ domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data we propose a model where the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.