Dissecting the structural and chemical determinants of the "open-to-closed" motion in the mannosyltransferase PimA from Mycobacteria

Ane Rodrigo-Unzueta, Mattia Ghirardello, Saioa Urresti, Ignacio Delso, David Giganti, Itxaso Anso, Beatriz Trastoy, Natalia Comino, Montse Tersa, Cecilia D'Angelo, Javier O. Cifuente, Alberto Marina, Jobst Liebau, Lena Mäler, Alexandre Chenal, David Albesa-Jové, Pedro Merino, Marcelo E. Guerin

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Abstract

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-To-β-strand and β-strand-To-α-helix transitions and (ii) an "open-To-closed"motion between the two Rossmann-fold domains, a conformational change that is 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 an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor 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 β-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 not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

Original languageEnglish
Pages (from-to)2934-2945
Number of pages12
JournalBiochemistry
Volume59
Issue number32
Early online date28 Jul 2020
DOIs
Publication statusPublished - 18 Aug 2020

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