Locking GTPases covalently in their functional states
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Locking GTPases covalently in their functional states. / Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Goody, Roger S.
in: NAT COMMUN, Jahrgang 6, 16.07.2015, S. 7773.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Locking GTPases covalently in their functional states
AU - Wiegandt, David
AU - Vieweg, Sophie
AU - Hofmann, Frank
AU - Koch, Daniel
AU - Li, Fu
AU - Wu, Yao-Wen
AU - Itzen, Aymelt
AU - Goody, Roger S
PY - 2015/7/16
Y1 - 2015/7/16
N2 - 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.
AB - 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.
KW - Binding Sites
KW - Crystallography, X-Ray
KW - Escherichia coli
KW - Escherichia coli Proteins
KW - Fungal Proteins
KW - GTP Phosphohydrolases
KW - Guanosine Diphosphate
KW - Guanosine Triphosphate
KW - Protein Binding
KW - rab GTP-Binding Proteins
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1038/ncomms8773
DO - 10.1038/ncomms8773
M3 - SCORING: Journal article
C2 - 26178622
VL - 6
SP - 7773
JO - NAT COMMUN
JF - NAT COMMUN
SN - 2041-1723
ER -