Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria

Abstract

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myoinositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) -helix-to--strand and -strand-to--helix transitions, and (ii) an 'open-to-closed' motion between the two Rossmann-fold domains, a conformational change necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such activation mechanism we analyzed a series of chemical derivatives, including mannose-phosphate (Man-P) and mannose-pyrophosphateribose (Man-PP-RIB), and additional GDP derivatives, as pyrophosphate-ribose (PP-RIB) and GMP, by the combined used of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry and Small Angle X-ray Scattering methods. Although the phosphate is present, we found that the mannose ring, neither covalently attached to phosphate (Man-P) nor to PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the 'open-to-closed' motion, with the -phosphate group providing the high affinity binding to PimA. Altogether, the experimental data, contribute to a better understanding of the structural determinants involved in the 'open-to-closed' motion observed not only in PimA, but also visualized/predicted in other glycosyltransfeases. In addition, the experimental data might prove useful for the discovery/development of PimA and/or glycosyltransferase inhibitors.

Understanding the Mannose Transfer Mechanism of Mycobacterial Phosphatidyl-myo-inositol Mannosyltransferase A from Molecular Dynamics Simulations

“…Further inspection of the values of this angle revealed two distinct populations of the angles where the open and closed conformations of PimA showed peaks at 115 and 145°, respectively ( Figure S4-B ). This result is apparently contrasting to a recent experimental work, 20 which showed that the nucleotide part and β-PO 4 2– of GDPM is responsible for facilitating “open to closed motion” of PimA, whereas our results show that PimA itself can fold back to the closed conformation. It may be possible that the “closed to open” motion of PimA is partially inhibited in the presence of GDPM due to its nucleotide moiety and β-PO 4 2 group.…”

“…The C-terminal residues 349–373 of PimA fold over to the N-terminal domain . Coordinating motions of these Rossman fold-like domains gave rise to one closed conformation and another open conformation, as revealed by small-angle X-ray scattering (SAXS) studies . The structure of PimA was also shown to go through secondary structure transitions while shuffling between closed and open conformations .…”

“… 6 Coordinating motions of these Rossman fold-like domains gave rise to one closed conformation and another open conformation, as revealed by small-angle X-ray scattering (SAXS) studies. 20 The structure of PimA was also shown to go through secondary structure transitions while shuffling between closed and open conformations. 18 It had also been shown that the presence of both the fatty acyl chains of PI is absolutely necessary for PimA to transfer the mannose group from GDPM to PI.…”

HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA

“…The decrease in red regions in the UDP–CgMshA structure relative to the unliganded MshA structure suggests that the UDP–CgMshA complex results in a decrease in dynamics for much of the core structure in both domains. This is consistent with the results of previous limited proteolysis experiments with PimA that show protection from degradation in the presence of the nucleotide portion of the donor substrate . External regions and mobile loops are less affected by the presence of UDP, and no regions of EX1 exchange were identified for this complex.…”

“…This is consistent with the results of previous limited proteolysis experiments with PimA that show protection from degradation in the presence of the nucleotide portion of the donor substrate. 10 External regions and mobile loops are less affected by the presence of UDP, and no regions of EX1 exchange were identified for this complex. The stark contrast between the overall decrease in exchange for the UDP−CgMshA product complex and the UDP−GlcNAc− CgMshA substrate complex suggests that the donor sugar ring plays an important role in activating/maintaining local dynamics in the closed structure.…”