Substrate-induced Conformational Changes in the Essential Peripheral Membrane-associated Mannosyltransferase PimA from Mycobacteria

Abstract

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), which are key components of the mycobacterial cell envelope. PimA is the paradigm of a large family of peripheral membrane-binding GTs for which the molecular mechanism of substrate/membrane recognition and catalysis is still unknown. Strong evidence is provided showing that PimA undergoes significant conformational changes upon substrate binding. Specifically, the binding of the donor GDP-Man triggered an important interdomain rearrangement that stabilized the enzyme and generated the binding site for the acceptor substrate, phosphatidyl-myo-inositol (PI). The interaction of PimA with the ␤-phosphate of GDP-Man was essential for this conformational change to occur. In contrast, binding of PI had the opposite effect, inducing the formation of a more relaxed complex with PimA. Interestingly, GDP-Man stabilized and PI destabilized PimA by a similar enthalpic amount, suggesting that they formed or disrupted an equivalent number of interactions within the PimA complexes. Furthermore, molecular docking and site-directed mutagenesis experiments provided novel insights into the architecture of the myo-inositol 1-phosphate binding site and the involvement of an essential amphiphatic ␣-helix in membrane binding. Altogether, our experimental data support a model wherein the flexibility and conformational transitions confer the adaptability of PimA to the donor and acceptor substrates, which seems to be of importance during catalysis. The proposed mechanism has implications for the comprehension of the peripheral membrane-binding GTs at the molecular level.Glycans are not only one of the major components of the cell but also are essential molecules that modulate a variety of important biological processes in all living organisms. Glycans are used primarily as energy storage and metabolic intermediates as well as being main structural constituents in bacteria and plants. Moreover, as a consequence of protein and lipid glycosylation, glycans generate a significant amount of structural diversity in biological systems. This structural information is particularly apparent in molecular recognition events including cell-cell interactions during critical steps of development, the immune response, host-pathogen interactions, and tumor cell metastasis. Most of the enzymes encoded in eukaryotic/prokaryotic/archaeans genomes that are responsible for the biosynthesis and modification of glycan structures are GTs 3(1). Here we have focused in the phosphatidyl-myo-inositol mannosyltransferase A (PimA), an essential enzyme of mycobacterial growth that initiates the biosynthetic pathway of key structural elements and virulence factors of Mycobacterium tuberculosis, the phosphatidyl-myo-inositol mannosides (PIM) lipomannan and lipoarabinomannan (2-5). This amphitropic enzyme catalyzes the transfer of a Manp residue from GDPMan to the 2-position of PI to form phosphatidyl...

Spatially distinct and metabolically active membrane domain in mycobacteria

“…S14), confirming the previous studies suggesting that AcPIM2 is produced in the PMf (12). The absence of the first mannosyltransferase PimA in the PMf proteome was expected because it is an amphipathic enzyme transiently associating with membrane by weak ionic interactions (12,45). Because AcPIM2 is found in both membranes, AcPIM2 produced in the PMf must be distributed to the PM-CW.…”

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

“…The double mutant variants of PimA, W82F/W154F, W82F/W349F, and W154F/W349F, were obtained from GenScript by using pET29a-pimA (14) as the DNA template and expressed and purified as described previously (14). The purified W82F/W154F, W82F/W349F, and W154F/W349F PimA variants displayed enzymatic activity, demonstrating that they are functional (Fig.…”

“…Materials and Methods-Recombinant PimA from Mycobacterium smegmatis was produced in Escherichia coli and purified to apparent homogeneity as described previously (10,11,14). The double mutant variants of PimA, W82F/W154F, W82F/W349F, and W154F/W349F, were obtained from GenScript by using pET29a-pimA (14) as the DNA template and expressed and purified as described previously (14).…”

“…However, its transferase activity was stimulated by diC8PI at a concentration of 300 M (i.e. five times the CMC) or high concentrations of non-substrate anionic surfactants, such as cardiolipin or 1,2-dipalmitoyl-sn-glycero-3-phosphate, but not with the zwitterion phosphatidylethanolamine or 1,2-dipalmitoylsn-glycerol, indicating that the reaction requires a lipid-water interface (11,14). 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).…”

“…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.…”