An intramolecular salt bridge drives the soluble domain of GTP-bound atlastin into the postfusion conformation

Abstract

Endoplasmic reticulum (ER) network branching requires homotypic tethering and fusion of tubules mediated by the atlastin (ATL) guanosine triphos-phatase (GTPase). Recent structural studies on the ATL soluble domain reveal two dimeric conformers proposed to correspond to a tethered prefusion state and a post-fusion state. How the prefusion conformer transitions to the postfusion conformer is unknown. In this paper, we identify an intramolecular salt bridge mediated by two residues outside the GTPase domain near the point of rotation that converts the prefusion dimer to the post-fusion state. Charge reversal of either residue blocked ER network branching, whereas a compensatory charge reversal to reestablish electrostatic attraction restored function. In vitro assays using the soluble domain revealed that the salt bridge was dispensable for GTP binding and hydrolysis but was required for forming the post-fusion dimer. Unexpectedly, the postfusion conformation of the soluble domain was achieved when bound to the nonhydrolyzable GTP analogue guanosine 5′-[β,γ-imido]triphosphate, suggesting that nucleotide hydrolysis might not be required for the prefusion to postfusion conformational change. © 2011 Morin-Leisk et al.