Structural basis for Arl3-specific release of myristoylated ciliary cargo from UNC119

Abstract

Access to the ciliary membrane for trans-membrane or membrane-associated proteins is a regulated process. Previously, we have shown that the closely homologous small G proteins Arl2 and Arl3 allosterically regulate prenylated cargo release from PDEd. UNC119/HRG4 is responsible for ciliary delivery of myristoylated cargo. Here, we show that although Arl3 and Arl2 bind UNC119 with similar affinities, only Arl3 allosterically displaces cargo by accelerating its release by three orders of magnitude. Crystal structures of Arl3 and Arl2 in complex with UNC119a reveal the molecular basis of specificity. Contrary to previous structures of GTP-bound Arf subfamily proteins, the N-terminal amphipathic helix of Arl3 GppNHp is not displaced by the interswitch toggle but remains bound on the surface of the protein. Opposite to the mechanism of cargo release on PDEd, this induces a widening of the myristoyl binding pocket. This leads us to propose that ciliary targeting of myristoylated proteins is not only dependent on nucleotide status but also on the cellular localization of Arl3.

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

“…As predicted from sequence signatures, 22 structural studies of Arf-like and Arf-related GTPases, including Golgi Arl1, 74,75 cilium Arl3 76,77,78 and Arl6 79 and endoplasmic reticulum Sar1 80,81 have now shown that autoinhibition by the N-terminal extension and the interswitch takes place in these GTPases and is released by the displacement of the N-terminus and the toggle of the interswitch. Thus, the allosteric mechanism that uses the displacement of the N-terminal extension as a priming event to autoinhibition release and the remodeling of the interswitch as the means whereby information is propagated to the nucleotide-binding site is probably general to the entire Arf-like family.…”

“…The N-termini of Arfrelated GTPases vary in length, sequence and post-translational modifications, and there is therefore ample reasons to believe that they are displaced in different ways to release autoinhibition. For instance, the N-terminus of Arl3-GTP interacts with another protein, 77,78,82 and it is conceivable that there exists Arf-like GTPases in which autoinhibition release involves interactions of the Nterminus with both a protein and a membrane. Understanding this process will be a major theme for future investigations of the molecular mechanisms that regulate the activity of Arf-like GTPases.…”

Sorting of lipidated cargo by the Arl2/Arl3 system

“…INPP5E showed an approximately 100 fold higher binding affinity to PDE6d with a dissociation constant in the nanomolar range as compared to submicromolar affinities for Ras and Rheb. 46 Stimulated by the findings that only Arl3 can release high affinity ciliary cargo from Unc119 12,15 and from PDE6d, 38 we could confirm that under identical concentrations only Arl3 is able to release a C-terminal prenylated INPP5E peptide from its complex with PDE6d, while Arl2 did not. 46 Kinetic studies showed that the stimulated release of farnesylated INPP5E peptide by Arl3 GTP is 600fold faster than by Arl2 GTP.…”

“…The binding of type 1 and type 2 effectors is nevertheless mutually exclusive. 8,9,[13][14][15] Functional differences between Arl2 and Arl3…”

“…The structure of a complex between Unc119a and Arl3 showed that Arl2/3 binding to Unc119 might be unfavorable for the binding of myristoylated cargo. This was deduced from a superimposition with the structure between a lauroylated peptide from GNAT1 (the a subunit of transducin) 15 and Unc119a (Fig. 2).…”