Adenyl ribosylation factor (Arf) is a small GTPase that lacks intrinsic GTPase activity and requires its GTPase-activating protein (ArfGAP) to function. ASAP1 is an ArfGAP composed of BAR, Pleckstrin Homology (PH), ArfGAP, ankyrin repeat, E/DLPPKP repeat and SH3 domains. The minimum catalytic unit of ASAP1—referred to as ASAP1-PZA—is composed of the PH domain and the catalytic ArfGAP domain (ZA domain). The ZA domain must interact directly with the GTP substrate for catalysis to occur and is connected to the PH domain via a flexible linker. Experimental NMR data indicate that the ZA domain predominantly adopts conformations unbound to ArfGTP. Mutations that lengthen the linker results in reduced catalytic activity. Relating the catalytic rate to linker structure requires molecular details that determine the stability of the ZA-ArfGTP bound and unbound states. Such a model should consider not only the conformational space allowed by the linker, but also the enthalpy of the bound and unbound states. AlphaFold predicts a key interaction between an arginine residue in the ZA domain and the phosphate group of GTP, presumably stabilizing the bound state and consistent with the residue’s role in catalysis. Metadynamics simulations show that the AlphaFold state is reformed after contacts are broken, but also indicates greater stability of the ArfGTP-ZA complex than that inferred from a straightforward interpretation of the NMR experiment. Simulations also show that the linker is not highly restricted in either the bound or unbound state, consistent with a model in which it only extends the unbound conformational volume. While challenging to converge, these calculations suggest that the favorability of the unbound state of the ZA domain cannot be explained solely by entropy.
Hu et al. (Sun,) studied this question.