Putting a lid on it: The N-terminal helix of Arf1 inhibits switching via uniform stabilization of the 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.