Fig. 2: Binding of ASAP1 PH to myrArf1 at the membrane.
From: An active allosteric mechanism in ASAP1-mediated Arf1 GTP hydrolysis redefines PH domain function
A Homology model of human myrArf118 is shown in grey ribbon format at the membrane surface. Switch 1 (residues 40–49, dark blue), switch 2 (residues 68–78, cyan) and the interswitch (residues 50–67, light blue) are highlighted. Residues 2–13 form the N-terminal helix (embedded in the membrane), and residues 17–181 constitute the G domain (solvent-exposed). Images created using Chimera56. B 1H-13C HMQC spectrum centered on the Ile region of 50 µM U-2H,15N, δ1-13C1H-labeled Ile, δ1/δ2 -13C1H-labeled Leu and γ1/γ2-13C1H-labeled Val myrArf1·GTP on a nanodisc (PC:PI(4,5)P2 = 95:5) in the absence (left, black) or in the presence of 2H ASAP1 PH (right, blue) at a Arf:PH ratio of 1:1.2). C 1H-13C CSPs observed between myrArf1 free or bound to ASAP1 PH domain (Arf:PH 1:1.2) plotted against residue number. When applicable, data are presented as the average of the pro-R and pro-S methyl groups. The error bars were calculated based on the digital resolution of the spectra, as described in “Methods”. D 1H-13C CSP values are mapped on the Arf1 surface: CSP > 2σ (red), CSP > 1σ (orange). Images created using Chimera56. E Ratio of intensities of Arf1 1H-13C methyl cross peaks measured in the presence of 1H ASAP1 and 2H ASAP1 PH plotted against residue number. When applicable, data are presented as the average of the pro-R and pro-S methyl groups. Error bars were calculated based on the signal-to-noise (S/N) ratio of the spectra as described in Methods. F Ratios are mapped on the Arf1 surface with I1H-PH/I2H-PH < 0.8 (dark blue) and 0.9 < I1H-PH/I2H-PH < 0.8 (light blue). In addition to strong effects observed for V53 and residues of switch 1 and 2, the small amount of broadening observed for methyl groups on the N-terminal of myrArf1 may indicate proximity of the membrane bound part of myrArf1 with the ASAP1 PH domain as proposed in Roy et al.4. Images created using Chimera56. Source data are provided as a Source data file.
