title: Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria.
abstract: Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes β-strand-to-α-helix and α-helix-to-β-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.
authors.affiliation: Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC, UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain,; Departamento de Bioquímica, Universidad del País Vasco, 48940 Leioa, Vizcaya, Spain.
authors.forename: Ane
authors.initials: A
authors.lastname: Rodrigo-Unzueta
authors.affiliation: Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 and University Paris Diderot, Sorbonne Paris Cité, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Mariano A
authors.initials: MA
authors.lastname: Martínez
authors.affiliation: Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC, UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain,; Departamento de Bioquímica, Universidad del País Vasco, 48940 Leioa, Vizcaya, Spain,; Structural Biology Unit, Center for Cooperative Research in Biosciences (CIC-bioGUNE), Bizkaia Technology Park, 48160 Derio, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Comino
authors.affiliation: Institut Pasteur, Unité de Microbiologie Structurale, CNRS UMR 3528 and University Paris Diderot, Sorbonne Paris Cité, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.affiliation: Unité de Biochimie des Interactions Macromoléculaires and CNRS UMR 3528, 28 Rue du Dr. Roux, 75724, Paris Cedex 15, France. Electronic address: alexandre.chenal@pasteur.fr.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
authors.affiliation: Unidad de Biofísica, Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco/Euskal Herriko Unibertsitatea (CSIC, UPV/EHU), Barrio Sarriena s/n, Leioa, Bizkaia 48940, Spain,; Departamento de Bioquímica, Universidad del País Vasco, 48940 Leioa, Vizcaya, Spain,; Structural Biology Unit, Center for Cooperative Research in Biosciences (CIC-bioGUNE), Bizkaia Technology Park, 48160 Derio, Spain,; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain. Electronic address: mrcguerin@cicbiogune.es.
authors.forename: Marcelo E
authors.initials: ME
authors.lastname: Guerin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D008364:Mannosyltransferases; C543796:phosphatidyl-myo-inositol mannosyltransferase PimA, Mycobacterium smegmatis
keywords: enzyme; glycolipid; glycosyltransferase; membrane enzyme; structure-function