High-pressure studies reveal the conformational landscape of the small GTPase Arf6.

Abstract

Arf6, a small GTPase, functions as a protein “switch” by coupling the binding and subsequent hydrolysis of GTP to a variety of signaling cascades within the cell. Despite having significant degrees of sequence and structural homology in both the GDP and GTP-bound forms to the more well-characterized protein Arf1, the two proteins exhibit distinct spatial and functional segregation: Arf1 is responsible for membrane trafficking between the Endoplasmic Reticulum and Golgi Apparatus, while Arf6 is responsible for trafficking between the plasma membrane and endosome. Furthermore, the accessory proteins required for in vivo functionality of the switch are not interchangeable. Arf proteins are known to undergo massive structural rearrangements during the nucleotide switch transition, especially in the N-terminal and switch regions. Thus, it is unlikely that Arf protein binding partners’ specificity stems purely from the end state structures. We hypothesize that the excited conformations sampled during this transition are responsible for the observed specificity. To characterize these excited conformational states, our lab utilizes pressure-induced perturbation techniques which locally unfold the protein. We gain insights into the state of the protein using a two-pronged approach consisting of both high-pressure NMR and high-pressure SAXS; this has allowed us to develop residue-specific unfolding curves for Arf6 as well as pair-distance distribution curves to understand the global shape of the protein at various pressures. Using these results, we have begun to structurally map the conformational landscape of Arf6.