Abstract
Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, 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 low resolution envelopes obtained from small angle x-ray 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 wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.
Highlights
Knowledge of conformational changes and dynamics occurring in glycosyltransferases is limited
Phosphatidyl-myo-inositol mannosyltransferase A (PimA) Displays an Intrinsic Low Mechanical Stability—Single molecule force spectroscopy has been proven to be a powerful technique to understand the role of force in mechanical stability and to study protein conformational dynamics and enzyme catalysis [43, 44] For instance, applying a calibrated force to the substrate of an enzyme can probe the dynamics of the active site during catalysis with subangstrom resolution [45]
The study of the conformational changes and dynamics that govern substrate recognition and catalysis remains a major challenge in the field of GTs
Summary
Knowledge of conformational changes and dynamics occurring in glycosyltransferases is limited. The PimA1⁄7GDP complexed forms clearly demonstrate that 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 wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane. The crystal structure of PimA from Mycobacterium smegmatis has been recently solved in complex with both the donor substrate GDPManp and GDP, one of the reaction products Both structures superimpose well (root mean square deviation of 0.3 Å for 361 identical residues) and reveal that the enzyme displays the typical GT-B fold of GTs, one of the two major structural folds described for the nucleotide-sugar-dependent enzymes (Ref. 14 and see Fig. 1A). In combination with the solution structure of PimA by small-angle X-ray scattering, we propose a model for substrate binding wherein protein flexibility and conformational transitions play a prominent role
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