Structural and Functional Studies of Ubiquitin Binding Domains 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. The main focus of this project in 2009 was to extend determine how the structure of ESCRT-0 influences its ability to bind a diverse set of ubiquitinated receptors. ESCRT-0 consists of the Hrs and STAM subunits, which are held together by a helical core. We determined the crystal structure of the human ESCRT-0 core. The Hrs subunit contains a DUIM ubiquitin binding domain, and the STAM subunit contains ubiquitin-binding VHS and UIM domains. The overall structure was characterized by a Monte Carlo-based simulation method that incorporated solution hydrodynamic data and the solved structures of the individual domains and the structural core. The model for the intact structure shows that it is highly flexible and suggests that this flexibility can be used to bind a wide range of cargoes of different sizes and ubiquitination states.
| Hurley, James H (2011) Nipped in the bud: how the AMSH MIT domain helps deubiquitinate lysosome-bound cargo. Structure 19:1033-5 |
| Hurley, James H; Stenmark, Harald (2011) Molecular mechanisms of ubiquitin-dependent membrane traffic. Annu Rev Biophys 40:119-42 |
| Ren, Xuefeng; Hurley, James H (2010) VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo. EMBO J 29:1045-54 |
| Wollert, Thomas; Hurley, James H (2010) Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464:864-9 |
| Li, Wei; Tu, Daqi; Li, Lianyun et al. (2009) Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2. Proc Natl Acad Sci U S A 106:3722-7 |
| Ren, Xuefeng; Kloer, Daniel P; Kim, Young C et al. (2009) Hybrid structural model of the complete human ESCRT-0 complex. Structure 17:406-16 |
