Conformational Plasticity of the Essential Membrane-associated Mannosyltransferase PimA from Mycobacteria

Abstract

Background: Knowledge of conformational changes and dynamics occurring in glycosyltransferases is limited. Results: PimA unfolds at low force, following heterogeneous multiple step mechanical unfolding pathways. Conclusion: With the elucidation of the solution structure of PimA, we conclude that the enzyme displays remarkable structural plasticity that seems to be important for substrate/membrane association. Significance: This work constitutes the first conformational study of a glycosyltransferase at the single molecule level.

The Phosphatidyl- myo -Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

“…structure of PimA in the presence of GDP or GDP-Man was disclosed in 2007 and represented the very first structure of a glycosyltransferase found to be involved in mycobacterial cell envelope biosynthesis (38). Overall, the availability of both structural and strong mechanistic information about PimA (19,39,40), along with its essentiality for in vitro growth in mycobacteria (33,41) and a lack of a human homolog, makes PimA an appealing drug target (42). Our goal in this study was to investigate the essentiality of M. tuberculosis PimA in a mouse model of TB in order to evaluate its potential for drug discovery purposes.…”

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria

“…The apo-extended conformation might account for an additional lobe observed in small angle x-ray scattering studies (Fig. 1B) (15). However, the most striking observation is the reshuffling of secondary structure elements on the N-terminal domain of the PimA-GDPMan complex (11,12).…”

“…First, the crystal and solution structures of PimA revealed several unprecedented conformational changes, including ␤-strandto-␣-helix and ␣-helix-to-␤-strand transitions, reflecting its inherent plasticity/variability. Second, single molecule force spectroscopy indicates that PimA not only exhibits weak mechanical stability but also unfolds the following heterogeneous multiple step mechanical unfolding pathways akin to MGlike states (15). Finally, the combination of near UV CD and thermally induced unfolding followed by CD and differential scanning calorimetry revealed (i) a loss of tertiary structure and (ii) a loss of the cooperativity during the thermally induced unfolding process in the presence of non-substrate anionic vesicles (herein and see Ref.…”

“…Trp-154 residue is located in a region of the N-terminal domain of PimA (residues 129 -163), exhibiting important conformational variability, including the reshuffling of secondary structure elements in the form of ␣-helix-to-␤-strand and ␤-strand-to-␣-helix transitions (12,15). The FIR 320/360 of the PimA-Trp-154 ratio of fluorescence intensities indicates that Trp-154 is exposed to a more apolar environment in the presence of DOPG or PI-containing SUVs (Table 3).…”

“…1B) (10 -13). This structural information revealed unprecedented conformational changes that appear to be critical during substrate/membrane binding and catalysis, including the reshuffling of secondary structural elements and interdomain (open-to-closed) motions associated with GDP-Man binding between the N-and C-terminal domains, which markedly stabilize the enzyme (12,14,15). Specifically, the nucleotide moiety of GDP-Man seems to be the main driving force to trigger the open-to-closed motion, with the ␤-PO 4 group playing a key role in the following: (i) providing the high affinity binding of the nucleotide sugar to PimA and (ii) the generation of a competent active site.…”