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

Rodrigo-Unzueta, Ane, Ghirardello, Mattia, Urresti, Saioa, Delso, Ignacio, Giganti, David, Anso, Itxaso, Trastoy, Beatriz, Comino, Natalia, Tersa, Montse, D'Angelo, Cecilia, Cifuente, Javier O., Marina, Alberto, Liebau, Jobst, Mäler, Lena, Chenal, Alexandre, Albesa-Jové, David, Merino, Pedro and Guerin, Marcelo E. (2020) Dissecting the structural and chemical determinants of the "open-to-closed" motion in the mannosyltransferase PimA from Mycobacteria. Biochemistry, 59 (32). pp. 2934-2945. ISSN 0006-2960

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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.

Item Type: Article
Additional Information: Funding Information: This work was supported by grants from the European Commission, New Medicine for Tuberculosis, Contract LSHP-CT-2005-018923, and More Medicines for Tuberculosis, Contract HEALTH-F3-2011-260872 (M.E.G.); MARIE CURIE Reintegration Contract 844905 (B.T.); MINECO/FEDER EU Contracts SAF2010-19096, BIO2013-49022-C2-2-R, and BFU2016-77427-C2-2-R; Severo Ochoa Excellence Accreditation SEV-2016-0644 (M.E.G.); MINECO/FEDER EU Contract PID2019-104090RB-100 (P.M.); and “Juan de la Cierva Postdoctoral program” Contract IJCI-2014-19206 (B.T.).
Uncontrolled Keywords: biochemistry,sdg 3 - good health and well-being ,/dk/atira/pure/subjectarea/asjc/1300/1303
Faculty \ School: Faculty of Science > School of Pharmacy
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Depositing User: LivePure Connector
Date Deposited: 25 May 2022 10:30
Last Modified: 21 Jun 2022 08:36
URI: https://ueaeprints.uea.ac.uk/id/eprint/85153
DOI: 10.1021/acs.biochem.0c00376

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