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: 14.02). 04/2009; 33(6):775-83. DOI: 10.1016/j.molcel.2009.02.011
Source: PubMed


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.
    Molecular cell 03/2015; 58(1). DOI:10.1016/j.molcel.2015.01.041 · 14.02 Impact Factor
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    • "The selectivity of Eps15 tUIM for binding different ubiquitin chains has not been reported. The distance between the two UIMs would fit the configuration of a K63-linked diubiquitin (Sims and Cohen, 2009), which is consistent with our finding. Of note, molecular modeling predicts that the K33-linked diubiquitin adopts an extended conformation (Fushman and Walker, 2010), similar to the K63-linked diubiquitin. "
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    ABSTRACT: Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the trans-Golgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGN-derived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitin-dependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type.
    Molecular cell 04/2014; 54(4). DOI:10.1016/j.molcel.2014.03.035 · 14.02 Impact Factor
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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(10). DOI:10.1155/2013/167576
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