Biology subjects

McCallum, S. A.

Publications and source records attributed to McCallum, S. A..

2 recordsLinked to original sources

bioRxiv2025

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. Secondary AbstractThe mechanism of the Arf GTPase nucleotide switch implicates a functional molten globule (MG) ensemble. Switching by the front side of the Arf proteins is controlled by sequences on the back side. Here, using high pressure (HP) NMR we map a bidirectional and continuous allosteric pathway that passes between front and back through the GDP ligand. We also demonstrate that the allosteric mechanism controls access to the functional MG state, rather than directly affecting the switching rates.

biophysics↗

bioRxiv2025

Putting a lid on it: The N-terminal helix of Arf1 inhibits switching via stabilization of the overall native state

The Arf (and Arf-like) GTPases, unlike all other Ras family GTPase members, exhibit a repressed conformation in the inactive, GDP-bound form. The N-terminal helix of Arf GTPases, which is missing in the other Ras family members, caps the switch region, confining it to this repressed state. Nucleotide exchange and activation involve a massive conformational change made possible by the dissociation of the N-terminal helix from the core of the protein. Spontaneous switching in Arfs is enhanced upon deletion of this helix. While the structural and functional role of the N-terminal helix in Arf proteins is well-known, the energetic basis for its effects have not been established. Here we mapped the local stability of the Arf1{Delta}17 variant, deleted for the N-terminal helix, using high pressure biophysical approaches and compared it to that of full-length Arf1. Deletion of the N-terminal helix decreased Arf1 stability across the entire structure. Thus, rather than imposing a specific structural pathway for repression, the N-terminal helix exercises global control of Arf1 stability to repress switching. Statement of SignificanceEnergetic mapping of the N-terminal deletion mutant of Arf1 using high pressure biophysical approaches reveals that this helix maintains the protein in the repressed state unique to Arf and Arf-like GTPases via overall stabilization of the native state rather than unique allosteric communication with the switch region.

biophysics↗