title: Structural mechanism for ubiquitinated-cargo recognition by the Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding proteins.
abstract: The Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding (GGA) proteins are clathrin adaptors that mediate the sorting of transmembrane-cargo molecules at the trans-Golgi network and endosomes. Cargo proteins can be directed into the GGA pathway by at least two different types of sorting signals: acidic cluster-dileucine motifs and covalent modification by ubiquitin. The latter modification is recognized by the GGAs through binding to their GAT [GGA and TOM (target of Myb)] domain. Here we report the crystal structure of the GAT domain of human GGA3 in a 1:1 complex with ubiquitin at 2.8-A resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices alpha1 and alpha2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met-231, Asp-244, Glu-246, Leu-247, Glu-250, and Leu-251. The GAT-binding surface on ubiquitin is a hydrophobic patch centered on Ile-44 that is also responsible for binding most other ubiquitin effectors. The ubiquitin-binding site observed in the crystal is distinct from the Rabaptin-5-binding site on helices alpha2 and alpha3 of the GAT domain. Mutational analysis and modeling of the ubiquitin-Rabaptin-5-GAT ternary complex indicates that ubiquitin and Rabaptin-5 can bind to the GAT domain at two different sites without any steric conflict. This ability highlights the GAT domain as a hub for interactions with multiple partners in trafficking.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Gali
authors.initials: G
authors.lastname: Prag
authors.forename: Sangho
authors.initials: S
authors.lastname: Lee
authors.forename: Rafael
authors.initials: R
authors.lastname: Mattera
authors.forename: Cecilia N
authors.initials: CN
authors.lastname: Arighi
authors.forename: Bridgette M
authors.initials: BM
authors.lastname: Beach
authors.forename: Juan S
authors.initials: JS
authors.lastname: Bonifacino
authors.forename: James H
authors.initials: JH
authors.lastname: Hurley
mesh_terms: D020727:ADP-Ribosylation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D046912:Multiprotein Complexes; C097227:RABEP1 protein, human; D011994:Recombinant Proteins; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; D020727:ADP-Ribosylation Factors