Protein: DP00250 (UniProt P01555)
Region: residues 19	to 212
Annotation: disorder to order (Structural transition)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
the isolated CTA1 polypeptide has a disordered conformation with minimal enzymatic activity against Gs or synthetic substrates at 37°C (Murayama et al., 1993; Ray et al., 2012). When CTA1-ARF interactions have been examined at 37°C, CTA1 and ARF are typically added together before incubation at 37°C (Murayama et al., 1993; Pande et al., 2007). This protocol allows ARF6 to stabilize the folded conformation of CTA1 initially present at low temperature, but it does not determine whether ARF6 can activate the disordered CTA1 subunit. In this paper we examined whether ARF6 can activate a disordered, as well as a structured, CTA1 polypeptide. A biochemical and biophysical analysis of CTA1 structure / function indicated that ARF6 cannot promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted the disordered, 37°C structure of CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6.
Due to its intrinsic thermal instability, heating CTA1 to 37°C resulted in a loss of both α-helical and βsheet structures (Fig. 2B). Concomitant with this loss of secondary structure, the percentage of CTA1 irregular structure shifted from 10 ± 3% at 10°C to 55 ± 2% at 37°C (Table 1). When ARF6/GTP was added to CTA1 before heating to 37°C, the toxin retained its full αhelical structure and a substantial amount of its β-sheet structure at 37°C (Fig. 2C, Table 1). 
The impact of ARF6/GTP on CTA1 α-helical and β-sheet structure thus represents a partial stabilization of the folded toxin, and this is sufficient to maintain folded CTA1 in a protease-resistant conformation (Fig. 1A).
Collectively, these results demonstrated ARF6/GTP can partially stabilize the folded conformation of CTA1 but cannot promote the refolding of disordered CTA1
The lipochaperone function of lipid rafts (Ray et al., 2012) could act in conjunction with ARF to place disordered CTA1 in a folded, active conformation. This possibility was examined by FTIR spectroscopy (Fig. 4). CTA1 was heated to 37°C for 15 minutes before exposure to lipid raft-mimicking LUVs. Measurements of toxin structure were then recorded after an additional 30 minutes at 37°C (Fig. 4A). In the presence of lipid raft LUVs, the disordered conformation of CTA1 gained both α-helical and β-sheet content (Table 1). An additional gain of β-sheet structure and a further loss of irregular structure were detected when the disordered CTA1 subunit was incubated with both lipid rafts and ARF6/GTP (Fig. 4B). Under this condition, CTA1 exhibited an ordered conformation that matched the structural content of the folded, 10°C toxin (Table 1)
Because CTA1 is disordered at 37°C and ARF6/GTP does not promote the refolding of disordered CTA1 (Fig. 2, Table 1), it is necessary for other host factors such as lipid rafts to participate in shifting CTA1 from a disordered conformation to a partially folded active state that is competent for functional interaction with ARFs. The gain-of-structure resulting from toxin-raft interactions allows ARF6/GTP to induce an additional conformational change (Fig. 4, Table 1) that presumably stabilizes displacement of the occluding CTA1 “activation loop” and consequently optimizes the enzymatic activity of CTA1 (Fig. 5A).