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

Abstract

The Arf and Arf-like GTPases, unlike all other Ras family GTPase members, exhibit a repressed conformation in their inactive, GDP-bound form. An important component of this autoinhibition is their N-terminal helix, which is missing in the other Ras family members. This helix caps a switch element called the interswitch, confining it to this repressed state. Activation by GDP/GTP exchange is primed by the dissociation of the N-terminal helix from the core of the protein, which precedes a massive conformational change and binding of GTP. An important unanswered question is how the energetics of Arf-GDP is remodeled at the initial step of activation, permitting the GDP/GTP reaction to proceed. In cells, the helix is displaced through interaction with a membrane, an effect that can be mimicked in solution by truncation mutants. Here, we used Arf1Δ17, a construct in which the N-terminal helix was deleted, to map the local stability of Arf1-GDP using high-pressure biophysical approaches, which we compared to that of full-length Arf1. Remarkably, deletion of the N-terminal helix decreased Arf1 stability across the entire structure. Thus, rather than imposing a specific allosteric pathway for repression, the N-terminal helix exercises global control of Arf1 stability to repress switching. This has important implications for understanding the energetics basis of the cooperation of membranes and guanine nucleotide exchange factors in Arf and Arf-like proteins activation.