Structural and Functional Studies of Ubiquitin Binding Domains[unreadable] [unreadable] [unreadable] The covalent modification of proteins by ubiquitination is a major regulatory mechanism of protein degradation and quality control, endocytosis, vesicular trafficking, cell-cycle control, stress response, DNA repair, growth factor signaling, transcription, gene silencing, and other areas of biology. A class of specific ubiquitin binding domains mediates most of the effects of protein ubiquitination. The known membership of this group has expanded rapidly and now includes at least sixteen domains. The structures of many of the complexes with monoubiquitin have been determined, revealing interactions with multiple surfaces on ubiquitin. Inroads into understanding polyubiquitin specificity have been made for two UBA domains, whose structures have been characterized in complex with Lys48-linked diubiquitin. Several ubiquitin binding domains, including the UIM, CUE, and A20 ZnF, promote autoubiquitination, which regulates the activity of proteins that contain them. At least one of these domains, the A20 ZnF, acts as a ubiquitin ligase by recruiting a ubiquitin:ubiquitin conjugating enzyme thiolester adduct in a process that depends on the ubiquitin-binding activity of the A20 ZnF. The affinities of the monoubiquitin binding interactions of these domains span a wide range, but are most commonly weak, with Kd > 100 mM. The weak interactions between individual domains and monoubiquitin are leveraged into physiologically relevant high affinity interactions via several mechanisms: ubiquitin polymerization, modification multiplicity, oligomerization of ubiquitinated proteins and binding domain proteins, tandem binding domains, binding domains with multiple ubiquitin binding sites, and cooperativity between ubiquitin binding and binding through other domains to phospholipids and small G-proteins. The long terms goals of this project are to 1) determine the structural features of ubiquitin and its binding domains that are involved in molecular recognition; 2) correlate structural features with functional properties of these proteins in trafficking; and 3) understand the mechanisms whereby low-affinity interactions between individual binding domains and ubiquitin moieties and leveraged into physiological recognition events.[unreadable] [unreadable] Progress in FY 2008[unreadable] [unreadable] The main focus of this project in 2008 was to extend previous studies on ubiquitin-binding yeast ESCRT complexes to humans. We determined the crystal structure of the ubiquitin and lipid-binding human ESCRT-II with a truncation in its GLUE domain, and used hydrodynamic analysis and the previously solved structure of the human ESCRT-II GLUE domain to reconstruct a model of the intact complex and its mode of binding to ubiquitinated cargo in membranes. This led to new insights into the targeting of ESCRT-II to endosomes, which depends not only on the GLUE domain, but also on the basic helix 0 of the VPS22 subunit.