Locking GTPases covalently in their functional states.
David Wiegandt, Sophie Vieweg, Frank Hofmann, Daniel Koch, Fu Li, Yao-Wen Wu, Aymelt Itzen, Matthias P Müller, Roger S Goody
Author Information
David Wiegandt: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Sophie Vieweg: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Frank Hofmann: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Daniel Koch: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Fu Li: 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.
Yao-Wen Wu: 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.
Aymelt Itzen: Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany. ORCID
Matthias P Müller: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Roger S Goody: Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
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.
Associated Data
PDB | 4PHF; 4PHG; 4PHH
References
Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5621-6
[PMID: 22411835]
Mol Cell. 2009 Dec 25;36(6):1060-72
[PMID: 20064470]
Proc Natl Acad Sci U S A. 2014 Feb 18;111(7):2572-7
[PMID: 24550285]
