Characterizing conformational landscapes of arf gtpases using high pressure

Abstract

May2025School of ScienceThe adenosine diphosphate (ADP) ribosylation factor (Arf) small guanosine triphosphate (GTP)ases function as molecular switches to activate signaling cascades that control membrane organization in eukaryotic cells. In Arf1, the GDP/GTP switch does not occur spontaneously but requires guanine nucleotide exchange factors (GEFs) and membranes. The related small GTPase Arf6, however, is able to undergo spontaneous nucleotide exchange. In both cases, exchange involves massive conformational changes, including disruption of the core β-sheet. To probe the switch mechanism, we coupled pressure perturbation with nuclear magnetic resonance (NMR), Fourier Transform infrared spectroscopy (FTIR), small-angle X-ray scattering (SAXS), fluorescence, and computation. For both proteins, pressure induced the formation of a classical molten globule (MG) ensemble, though Arf6 populates an ensemble which is energetically distinct from that of Arf1. Pressure also favored the GDP to GTP transition in both proteins, providing strong support for the notion that the MG ensemble plays a functional role in the nucleotide switch. We propose that the MG ensemble allows for switching without the requirement for complete unfolding and may be recognized by GEFs. Substitutions in helix α5 of Arf6 locally destabilize this helix relative to Arf1, in which it is the most stable element. Mutation of the α5 sequence in Arf6 to that of Arf1 resulted in both increased stability and slower switching, demonstrating “back-to-front” control of nucleotide exchange kinetics. Evolutionary covariance analysis highlighted an extensive non-interacting coupling network in the C-terminal half of Arf6 as well as many other small GTPases. Our work suggests that an MG-based switching mechanism as well as “back-to-front” control of switching could constitute pervasive features in Arfs and Arf-like GTPases, and more generally, the evolutionarily related Rags (Ras-like small GTPases) and Gα GTPases.Ph