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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 [alpha]1 and [alpha]2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Glu-230, Met231, 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 [alpha]2 and [alpha]3 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. protein-protein interactions | structural biology | trafficking

The genetic and physiologic role played by SET domain enzymes is examined using a Rubisco large subunit methyltransferase-S-adenosylhomocysteine and a HEPES ion complex. Results show an all-beta structure for the SET domain whose conserved regions bind S-adenosylmethionine and lysine substrate. The catalysis is mediated by amino acid tyrosine at the conserved site.

Results show that VHS domain containing human target of myb 1 proteins are localized to membranes. Crystallographic analysis of VHS domains indicates that helicies 2 and 4 are involved in the protein-protein interaction.

The Golgi-localized, γ-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 EGA 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-Å resolution. Ubiquitin binds to a hydrophobic and acidic patch on helices α1 and α2 of the GAT three-helix bundle that includes Asn-223, Leu-227, Elu-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 lle-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 α2 and α3 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. [ABSTRACT FROM AUTHOR]