Book of Abstracts: Albany 2007
June 19-23 2007
NMR studies of the structure, dynamics, and ligand interactions of polyubiquitin chains provide clues to functional diversity of polyubiquitin
A variety of cellular processes are regulated by (poly)ubiquitin-mediated signaling events. Knowledge of the conformational properties of polyubiquitin chains is essential for the understanding of structural determinants of the functional diversity and linkage-specificity in polyubiquitin signaling(1). Our NMR studies of di- and tetra-ubiquitin in solution provide structural evidence that polyubiquitin?s conformation is linkage dependent (2, 3). Specifically, di-ubiquitin chains linked via Lys63 adopt an extended conformation, with no direct contact between the hydrophobic surfaces on the ubiquitins. In contrast, Lys48-linked di-ubiquitin forms a compact conformation, with a well-defined interface formed by the hydrophobic surface residues Leu8, Ile44, Val70 of the interacting ubiquitin units; similar contacts are observed in tetra-ubiquitin in solution and in the crystal(2, 4). This conformation, however, in not rigidly locked: the chain is in dynamic equilibrium between a ?closed? and one or more ?open? conformations (2). We present here a detailed picture of the process of opening/closing of the chain, derived from NMR relaxation measurements, and discuss the effect of pH and selected point mutations at the interface on the chain?s conformation(5, 6). The linkage-dependent conformational features and the dynamic nature of polyubiquitin chains have important implications for their interactions with various downstream cellular effectors, most of which bind directly to ubiquitin?s hydrophobic surface. Thus, ubiquitin units in a Lys63-linked chain are capable of binding ligands (for example, UBA2 domain of hHR23A) independently and in a mode similar to that for monoubiquitin(3). In contrast, ligand binding to Lys48-linked chains requires interface opening and could allow formation of a sandwich-like complex, as observed in the case of the UBA2:di-ubiquitin interaction(7). The conformational flexibility of Lys48-linked chains is critical for their ability to bind ligands. For example, restricting the opening of di-ubiquitin by its cyclization greatly reduced the chain?s ability to bind the UBA domain in this high-affinity mode(8). Similarly, the interface opening is involved in di-ubiquitin?s binding to the UIM-2 motif of the proteasomal subunit S5a. However, unlike the hHR23A UBA2 domain, two UIMs bind per Lys48-linked di-ubiquitin, and with similar affinity to that for Lys63-linked chain(9). We also discuss experimental data on polyubiquitin interactions with other ubiquitin-binding molecules in support of these predictions. Taken together, these studies provide structural insights into the relationship between linkage-dependent conformation of a polyubiquitin chain and the ability of differently linked chains to bind in a distinctive manner to effector proteins.
Supported by the NIH grant GM065334.
References and Footnotes
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA