Fig. 1: Binding of ASAP1 PH to myrArf1 is key for maximal GTP hydrolysis.
From: An active allosteric mechanism in ASAP1-mediated Arf1 GTP hydrolysis redefines PH domain function
A Top. The ArfGAP ASAP1 terminates Arf signaling by catalyzing GTP hydrolysis. Bottom. Schematic of ASAP1 and ASAP1 catalytic unit. The domain structure of ASAP1 is shown in the schematic at top. BAR Bin/amphiphysin/RVS, PH pleckstrin homology, Arf GAP Arf GTPase-activating protein, ANK ankyrin repeat, Pro-Rich proline-rich, (E/DLPPKP)8 tandem repeats of E/DLPPKP, SH3 Src homology 3. Recombinant proteins used in the studies are shown below the schematic of full-length ASAP1. The acronyms for the proteins include “P” for the PH domain, “Z” for the Arf GAP domain, which is a zinc-binding motif and “A” for the ankyrin repeat. The catalytic arginine Arg497 is part of the Z domain. B Schematic of known passive mechanisms by which PH domains affect protein function. C (left). Comparison of GAP activity using Arf1 as substrate in the presence of PI(4,5)P2 containing membranes. wt PZA (red circle), PHdZA (maroon ▲) or ZA (blue ■) was titrated into a GAP reaction containing 1 μM full-length Arf1 and LUVs at a total phospholipid concentration of 0.5 mM containing 5% mol PI(4,5)P2. (middle). Comparison of GAP activity using Arf1 as substrate in the presence of membranes without PI(4,5)P2 for wt PZA (red circle) and PHdZA (maroon open triangle). (right). Constructs design used in this study (see also Fig. SI1A). D Our approach to directly probe the role of the ASAP1 PH domain: comparing GAP activity using tandem constructs and isolated domains (“in trans” experiment). E The percentage of GTP bound to myrArf1 hydrolyzed in 3 min is plotted against the concentration of PZA (●), ZA (■) or ZA in the presence of 5 μM PH domain (▼). Source data are provided as a Source data file.
