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.
Highlights
GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states
We describe here the synthesis of reactive acryl derivatives of guanine nucleotides that are able to react covalently with strategically placed cysteines in GTPases to lock them into their functional states in an irreversible manner
The results described so far indicate that the conformations and properties of Rab1b are retained in a nucleotide-dependent manner in the covalent adducts with aGDP, aGTP or aGppNHp
Summary
GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. Proteins of the Ras superfamily of small GTPases regulate a variety of cellular processes such as intracellular transport, cell shape and motility, as well as differentiation and growth[1] For all of these processes, switching between the active GTP-bound and the inactive GDP-bound states is of fundamental importance[2]. Nucleotide analogues mimicking GDP or GTP may potentially exchange against the endogenous natural nucleotides in in vivo experiments, or specific GTPase amino acid substitutions may affect the molecular properties in a more complex and non-predictable manner than intended[6,7] The latter applies to the targeted GDP state, and there is essentially no method available for generating a conformation that is a true analogue of GTPase:GDP complexes without compromising other fundamental properties of the proteins (for example, binding to GEFs)[8]. In the case of Rab proteins, we show that the covalent adducts can be used to answer questions concerning the mechanism of localization to specific intracellular membranes
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