Molecular Interactions of Ubiquitin and Polyubiquitin with Ubiquitin Binding Domains
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Ubiquitin is a small protein that is covalently attached to proteins, either as a single ubiquitin moiety or as polyubiquitin chains. A cascade of enzymatic reactions is required for the isopeptide linkage between the C-terminus of ubiquitin and a lysine residue on a substrate protein or another ubiquitin. Attachment of ubiquitin or polyubiquitin, termed ubiquitination, mediates numerous cellular processes by acting as a versatile signal. The signal transmitted by the tag depends on the nature of the modification, which defines the specificity of the tag for different cellular machinery. This versatility is conferred by the variations in polyubiquitin tags, both in terms of length and lysine-linkage. Polyubiquitin chains can adopt a variety of different conformations based on these variations. The conformational and dynamic properties of the tag may optimize its binding to specific ubiquitin binding domains, therefore committing the target protein to distinct cellular outcomes.
A combination of NMR methods are used to study the interaction of several ubiquitin binding domains with Lys48- and Lys63-linked di-ubiquitin, the simplest model of a polyubiquitin chain, to gain insights into polyubiquitin recognition. The di-ubiquitin binding interface with ubiquitin-interacting motifs (UIMs) and ubiquitin-associated domains (UBAs) are mapped. Structural models of the complexes are also presented. The results provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interface. The results also show that the UBA domain of Ede1 preferentially binds to Lys63-linked di-ubiquitin. Structural models of the UBA in complex with Lys48- and Lys63-linked di-ubiquitin are shown.
Although ubiquitin is highly conserved in eukaryotes, it is promiscuous with regard to its binding partners, ranging from small molecules to UIM and UBA domains. This study examines the effects of point core leucine to serine mutations on UIM and UBA binding specificity. The results show that these mutations bestow ubiquitin with the ability to discriminate between ubiquitin-receptor proteins. Here, we solved the three-dimensional structure of the L69S Ub mutant in solution by NMR. These mutations have a profound effect on binding specificity while causing subtle changes in the protein's three-dimensional fold and reducing its stability.
Modification of a specific lysine located on Ub's hydrophobic surface has been reported to inhibit proteasomal degradation and endocytosis. Here, the effects of mutation to tryptophan at this position are investigated within the context of binding to a proteasomal receptor protein, hHR23A, and an endocytic receptor protein, Ede1.