Mapping Bidirectional Allosteric Communication in Arf GTPases

The Arf GTPases are eukaryotic signaling proteins implicated in trafficking, motility and membrane remodeling. They must undergo a massive conformational transition in the switch between their inactive, GDP-bound form and their GTP-bound form, competent for downstream signaling. The mechanism of their GDP-to-GTP nucleotide switch implicates a functional molten globule (MG) ensemble. Access to this ensemble and apparent spontaneous switching is modulated by residues in the C-terminal half of the different homologs through a back-to-front allosteric pathway. Here, using high pressure (HP) NMR we show that a mutation in the N-terminal switch region in the front of Arf1 known to modulate spontaneous switching perturbs the stability of residues on a path that stretches from the front to the back. This establishes the existence of a bidirectional and continuous allosteric pathway that passes through the GDP ligand. HP switching studies also demonstrate that the allosteric mechanism controls access to the functional MG state, rather than directly affecting the switching rates.