Abstract
SummaryUbiquitylation, the posttranslational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. The SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. Here, we present a structural model of the KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography. Remarkably, CUE1, a canonical CUE domain, recognizes a cluster of exposed hydrophobic and surrounding charged/amphipathic residues on KAP1, which are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data suggest that CUE domains may have a wider function than simply recognizing ubiquitin and the ubiquitin-fold.
Highlights
CUE domains are ubiquitin-binding domains (UBDs) that interact with ubiquitin (Ub) by occupying the hydrophobic pocket centered on the highly conserved Ub I44 residue (Hicke et al, 2005; Hofmann, 2009; Husnjak and Dikic, 2012)
SMARCAD1 and KAP1 Interact Directly in a Ubiquitylation-Independent Manner To study the tandem CUE domains of SMARCAD1 (Figure S1A), we generated human cell lines, depleted of endogenous SMARCAD1 by small hairpin RNA knockdown, which were reconstituted with a doxycycline-inducible, shRNA-resistant gene encoding either FLAG-tagged wild-type SMARCAD1 or SMARCAD1 with point mutations in the CUE domains (‘‘CUE1mt,2mt’’) (Figure S1B)
The CUE1mt,2mt possesses a total of eight alanine substitutions in the conserved, hydrophobic MFP and LL motifs (FP/AA and LL/LK/AA) that are important for Ub interaction
Summary
CUE domains are ubiquitin-binding domains (UBDs) that interact with ubiquitin (Ub) by occupying the hydrophobic pocket centered on the highly conserved Ub I44 residue (Hicke et al, 2005; Hofmann, 2009; Husnjak and Dikic, 2012). CUE domains have two main conserved sequence elements, a methionine-phenylalanine-proline motif (‘‘MFP’’; sometimes ‘‘hFP,’’ or even ‘‘haP,’’ where ‘‘h’’ indicates hydrophobic, and ‘‘a’’ aromatic residues), and a di-leucine repeat Where ‘‘i’’ indicates aliphatic, and ‘‘h’’ hydrophobic residues), both of which are essential for Ub binding (Kang et al, 2003; Ponting, 2000; Prag et al, 2003). Mutation of the UBD of a protein to perturb its interaction with Ub is often a reasonable starting point for interrogating the biological function of that protein
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