Structure of Arf6-GDP suggests a basis for guanine nucleotide exchange factors specificity

Arf6 is an isoform of Arf that localizes at the periphery of the cell where it has an essential role in endocytotic pathways. Its function does not ov erlap with that of Arf1, although the two proteins share similar to 70% seq uence identity and they have switch regions, whose conformation depends on the nature of the guanine nucleotide, with almost identical sequences. The crystal structure of Arf6-GDP at 2.3 Angstrom shows that it has a conformat ion similar to that of Arf1-GDP, which cannot bind membranes with high affi nity. Significantly, the switch regions of Arf6 deviate by 2-5 Angstrom fro m those of Arf1. These differences are a consequence of the shorter N-termi nal linker of Arf6 and of discrete sequence changes between Arf6 and Arf1. Mutational analysis shows that one of the positions which differs between A rf1 and Arf6 affects the configuration of the nucleotide binding site and t hus the nucleotide binding properties of the Arf variant Altogether, our re sults provide a structural basis for understanding how Arf1 and Arf6 can be distinguished by their guanine nucleotide exchange factors and suggest a m odel for the nudeotide/membrane cycle of Arf6.