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

DOI: 10.1021/acs.biochem.0c00376 DOI Help

Authors: Ane Rodrigo-unzueta (Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU)) , Mattia Ghirardello (University of Zaragoza-CSIC) , Saioa Urresti (Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU)) , Ignacio Delso (University of Zaragoza-CSIC) , David Giganti (University of Zaragoza-CSIC; Unité de Microbiologie Structurale (CNRS URA 2185), Institut Pasteur) , Itxaso Anso (CIC bioGUNE) , Beatriz Trastoy (CIC bioGUNE) , Natalia Comino (CIC bioGUNE) , Montse Tersa (CIC bioGUNE) , Cecilia D'angelo (CIC BioGUNE) , Javier O. Cifuente (CIC bioGUNE) , Alberto Marina (CIC bioGUNE) , Jobst Liebau (Stockholm University) , Lena Mäler (Stockholm University; Umeå University) , Alexandre Chenal (Unité de Biochimie des Interactions Macromoléculaires (CNRS UMR 3528), Institut Pasteur) , David Albesa-jove (Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU); CIC bioGUNE; IKERBASQUE, Basque Foundation for Science) , Pedro Merino (University of Zaragoza) , Marcelo E. Guerin (Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC,UPV/EHU); CIC bioGUNE; IKERBASQUE, Basque Foundation for Science)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry

State: Published (Approved)
Published: July 2020

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 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-pyrophosphate-ribose (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 β-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 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 observed not only in PimA, but also visualized/predicted in other glycosyltransferases. In addition, the experimental data might prove useful for the discovery/development of PimA and/or glycosyltransferase inhibitors.

Journal Keywords: carbohydrate-modifying enzyme; glycosyltransferase; conformational changes; structural biology; X-ray crystallography

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: B21-High Throughput SAXS

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