title: X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism.
abstract: Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.
authors.affiliation: 1] Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Av. República, EAN, 2780-157 Oeiras, Portugal [2].
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Nogly
authors.affiliation: 1] Université Grenoble Alpes, IBS, F-38000 Grenoble, France [2] CNRS, IBS, F-38000 Grenoble, France [3] CEA, IBS, F-38000 Grenoble, France [4] Interdisciplinary Center for Basic Research, Moscow Institute of Physics and Technology, 141700 Dolgoprudniy, Russia [5].
authors.forename: Ivan
authors.initials: I
authors.lastname: Gushchin
authors.affiliation: 1] Université Grenoble Alpes, IBS, F-38000 Grenoble, France [2] CNRS, IBS, F-38000 Grenoble, France [3] CEA, IBS, F-38000 Grenoble, France [4].
authors.forename: Alina
authors.initials: A
authors.lastname: Remeeva
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Av. República, EAN, 2780-157 Oeiras, Portugal.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Esteves
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Av. República, EAN, 2780-157 Oeiras, Portugal.
authors.forename: Nuno
authors.initials: N
authors.lastname: Borges
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Av. República, EAN, 2780-157 Oeiras, Portugal.
authors.forename: Pikyee
authors.initials: P
authors.lastname: Ma
authors.affiliation: 1] Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, 52425 Juelich, Germany [2] Institute of Crystallography, University of Aachen (RWTH), 52056 Aachen, Germany.
authors.forename: Andrii
authors.initials: A
authors.lastname: Ishchenko
authors.affiliation: 1] University Grenoble Alpes, LJK, F-38000 Grenoble, France [2] CNRS, LJK, F-38000 Grenoble, France [3] Inria, F-38000 Grenoble, France.
authors.forename: Sergei
authors.initials: S
authors.lastname: Grudinin
authors.affiliation: 1] Université Grenoble Alpes, IBS, F-38000 Grenoble, France [2] CNRS, IBS, F-38000 Grenoble, France [3] CEA, IBS, F-38000 Grenoble, France [4] Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, 52425 Juelich, Germany.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Round
authors.affiliation: 1] Department of Life Sciences, Imperial College London, London SW7 2AZ, UK [2] Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton OX11 0DE, UK [3] Research Complex at Harwell Rutherford, Appleton Laboratory, Harwell, Oxford, Didcot OX11 0FA, UK.
authors.forename: Isabel
authors.initials: I
authors.lastname: Moraes
authors.affiliation: 1] Interdisciplinary Center for Basic Research, Moscow Institute of Physics and Technology, 141700 Dolgoprudniy, Russia [2] Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, 52425 Juelich, Germany.
authors.forename: Valentin
authors.initials: V
authors.lastname: Borshchevskiy
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Av. República, EAN, 2780-157 Oeiras, Portugal.
authors.forename: Helena
authors.initials: H
authors.lastname: Santos
authors.affiliation: 1] Université Grenoble Alpes, IBS, F-38000 Grenoble, France [2] CNRS, IBS, F-38000 Grenoble, France [3] CEA, IBS, F-38000 Grenoble, France [4] Interdisciplinary Center for Basic Research, Moscow Institute of Physics and Technology, 141700 Dolgoprudniy, Russia [5] Institute of Complex Systems (ICS), ICS-6: Structural Biochemistry, Research Centre Juelich, 52425 Juelich, Germany.
authors.forename: Valentin
authors.initials: V
authors.lastname: Gordeliy
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa (ITQB-UNL), Av. República, EAN, 2780-157 Oeiras, Portugal.
authors.forename: Margarida
authors.initials: M
authors.lastname: Archer
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D003565:Cytidine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D019843:Archaeal Proteins; D003565:Cytidine Diphosphate; D010770:Phosphotransferases; D008274:Magnesium