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Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria
journal contribution
posted on 2020-08-04, 18:12 authored by 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. GuerinThe 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.