Defining the role of the N-terminal helix in the Arf1 nucleotide switch transition using pressure perturbation.

Abstract

ADP-Ribosylation Factors (Arfs) are small GTPases that act as molecular “switches” for the signaling cascades responsible for membrane organization within eukaryotic cells. Overactivation and mutations of Arfs or their binding partners have been implicated in several genetic neurological diseases and breast cancer. Within the Arf family, Arf1 is one of the most studied. In its inactive conformation, the protein is bound to GDP and requires the reorganization of the N-terminal third of the protein to accommodate the dissociation and subsequent bind of GTP. During this conformational change, the myristoylated N-terminal helix becomes disordered allowing Arf1 to associate with membranes. Previous literature suggests that the position of the N-terminal helix is the determinant of the nucleotide exchange. In the Δ17Arf1 mutant, increased structural dynamics is observed and the protein can undergo a switch transition in absence of a membrane. We hypothesize that the N-terminal helix of Arf1 enhances the stability of the GDP bound state of the protein and the nucleotide exchange occurs under stringent control. We used high-pressure 1H-15N HSQC NMR spectroscopy and high-pressure SAXS to investigate the effect of the N-terminal helix for protein stability and the nucleotide switch transition. Further, analysis of the relaxation rates from CPMG and CEST provide greater insight into the structural dynamics of the switch transition. Our data provides evidence that the stability of Arf1 can be attributed in part to the presence of the N-terminal helix.