title: Locking GTPases covalently in their functional states.
abstract: GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase-acryl-nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Wiegandt
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Sophie
authors.initials: S
authors.lastname: Vieweg
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Hofmann
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Koch
authors.affiliation: 1] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany [2] Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany.
authors.forename: Fu
authors.initials: F
authors.lastname: Li
authors.affiliation: 1] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany [2] Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany.
authors.forename: Yao-Wen
authors.initials: YW
authors.lastname: Wu
authors.affiliation: Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
authors.forename: Aymelt
authors.identifier: 0000000242495617
authors.initials: A
authors.lastname: Itzen
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Müller
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
authors.forename: Roger S
authors.initials: RS
authors.lastname: Goody
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020691:rab GTP-Binding Proteins