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 overlap with that of Arf1, although the two proteins share approximately 70% sequence 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 A shows that it has a conformation similar to that of Arf1-GDP, which cannot bind membranes with high affinity. Significantly, the switch regions of Arf6 deviate by 2-5 A from those of Arf1. These differences are a consequence of the shorter N-terminal linker of Arf6 and of discrete sequence changes between Arf6 and Arf1. Mutational analysis shows that one of the positions which differs between Arf1 and Arf6 affects the configuration of the nucleotide binding site and thus the nucleotide binding properties of the Arf variant. Altogether, our results provide a structural basis for understanding how Arf1 and Arf6 can be distinguished by their guanine nucleotide exchange factors and suggest a model for the nucleotide/membrane cycle of Arf6.
Ménétrey
J
J
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France.
Macia
E
E
Pasqualato
S
S
Franco
M
M
Cherfils
J
J
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000089982:ADP-Ribosylation Factor 6; D020033:Protein Isoforms; D007532:Isoleucine; D006153:Guanosine Diphosphate; D012694:Serine; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human