Linkage-Specific Avidity Defines the Lysine 63-Linked Polyubiquitin-Binding Preference of Rap80

Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
Molecular cell (Impact Factor: 14.46). 04/2009; 33(6):775-83. DOI: 10.1016/j.molcel.2009.02.011
Source: PubMed

ABSTRACT Linkage-specific polyubiquitin recognition is thought to make possible the diverse set of functional outcomes associated with ubiquitination. Thus far, mechanistic insight into this selectivity has been largely limited to single domains that preferentially bind to lysine 48-linked polyubiquitin (K48-polyUb) in isolation. Here, we propose a mechanism, linkage-specific avidity, in which multiple ubiquitin-binding domains are arranged in space so that simultaneous, high-affinity interactions are optimum with one polyUb linkage but unfavorable or impossible with other polyUb topologies and monoUb. Our model is human Rap80, which contains tandem ubiquitin interacting motifs (UIMs) that bind to K63-polyUb at DNA double-strand breaks. We show how the sequence between the Rap80 UIMs positions the domains for efficient avid binding across a single K63 linkage, thus defining selectivity. We also demonstrate K48-specific avidity in a different protein, ataxin-3. Using tandem UIMs, we establish the general principles governing polyUb linkage selectivity and affinity in multivalent ubiquitin receptors.

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    • "See also Figure S1. (Sims and Cohen, 2009). We first analyzed the linkage preference of the proteasome shuttling protein RAD23B that has been well characterized as a K48 binding protein (Rao and Sastry, 2002; Varadan et al., 2005). "
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    ABSTRACT: Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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    • "In addition, many UBDs have been shown to exhibit selectivity for certain types of polyubiquitin chains [7] [11] [13]. The linkage selectivity may also arise from multivalent binding between tandem UBD arrays in a given protein and ubiquitin monomers or linkages in a polyubiquitin chain [34] [35]. The function of tandem UBD arrays has been recently hypothesized to increase the affinity for a given ubiquitinated substrate rather than simultaneously engaging multiple substrates. "
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    ABSTRACT: Protein modifications by the covalent linkage of ubiquitin have significant involvement in many cellular processes, including stress response, oncogenesis, viral infection, transcription, protein turnover, organelle biogenesis, DNA repair, cellular differentiation, and cell cycle control. We provide a brief overview of the fundamentals of the regulation of protein turnover by the ubiquitin-proteasome pathway and discuss new therapeutic strategies that aim to mitigate the deleterious effects of its dysregulation in cancer and other human disease pathophysiology.
    08/2013; 2013. DOI:10.1155/2013/167576
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    • "Protein Expression and Enzymatic Synthesis of K11-Ub 2 Ub monomers were expressed and purified as described in Varadan et al., 2004; hHR23A UBA2 as in Varadan et al., 2004; UQ1-UBA as in Zhang et al., 2008; and Rap80 tUIM as in Sims and Cohen, 2009. PolyUb chains were made using controlled-length chain assembly (Pickart and Raasi, 2005) combined with domain-specific isotope labeling (Varadan et al., 2002). "
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    ABSTRACT: K11-linked polyubiquitin chains play important signaling and regulatory roles in both degradative and nonproteolytic pathways in eukaryotes. To understand the structural basis of how these chains are recognized and distinguished from other polyubiquitins, we determined solution structures of K11-linked diubiquitin (K11-Ub2) in the absence and presence of salt. These structures reveal that K11-Ub2 adopts conformations distinct from those of K48-linked or K63-linked chains. Importantly, our solution NMR and SANS data are inconsistent with published crystal structures of K11-Ub2. We found that increasing salt concentration compacts K11-Ub2 and strengthens interactions between the two Ub units. Binding studies indicate that K11-Ub2 interacts with ubiquitin-receptor proteins from both proteasomal and nonproteasomal pathways but with intermediate affinity and different binding modes than either K48-linked or K63-linked diubiquitin. Our data support the hypothesis that polyubiquitin chains of different linkages possess unique conformational and dynamical properties, allowing them to be recognized differently by downstream receptor proteins.
    Structure 07/2013; 21(7):1168-81. DOI:10.1016/j.str.2013.04.029 · 6.79 Impact Factor
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