Interactions of NEMO with diubiquitins: both revelations and confusions

NEMO is the regulatory subunit of the IKK (IκB kinase) complex for NF-κB activation. Recent studies have shown that the UBAN (ubiquitin binding in ABIN [A20-binding inhibitor of NF-κB activation] and NEMO [NF-κB essential modulator]) domain directly interacts with linear polyubiquitin chains [4-7]. The UBAN motif of NEMO forms a dimeric coiled coil and uses both chains for linear diubiquitin interactions. Structure-based mutagenesis and NMR studies revealed that the Ile44 patch and the C-terminal tail of the distal ubiquitin in linear diubiquitin are most critical for UBAN interaction [6]. The crystal structure of the UBAN-linear diubiquitin complex revealed interfaces that are similar to the NMR-based studies [7]. However, despite the measured stoichiometry in solution of one UBAN dimer per linear diubiquitin [6,8], the crystal structure showed that two dibubiquitin molecules interact symmetrically on each side of the UBAN dimer [7] (Figure 1B). It is possible that only one side of the dimeric UBAN is bound with diubiquitin in solution and that the other side was forced to interact with another diubiquitin molecule under the crystallization condition. Consistent with this assessment, it appears that at high diubiquitin concentrations, there is a second, weaker binding site [8].

The UBAN motif of NEMO also directly interacts with Lys63-linked polyubiquitin [4-6,9], albeit at a much lower affinity than with linear diubiquitin [6]. Similar mutagenesis and NMR-based methods revealed that the Ile44 patch of the proximal ubiquitin in Lys63-linked diubiquitin and the C-terminal tail of the distal ubiquitin provide the critical binding energy for the UBAN interaction [6]. In addition, the stoichiometry of the interaction was shown to be one UBAN dimer per Lys63-linked diubiquitin [6]. In contrast to the crystal structure of the UBAN-linear diubiquitin complex [7], the crystal structure of the UBAN-Lys63-linked diubiquitin complex showed that the diubiquitin interacts with only one side of the UBAN dimer [9] (Figure 1C). In fact, only one ubiquitin molecule in the diubiquitin, either the proximal or the distal ubiquitin, interacts with UBAN in the crystal lattice, and this is different from the higher energetic role of the proximal ubiquitin shown by mutagenesis experiments in solution [6].

Recent NMR and cellular studies showed that the C-terminal ZF of NEMO is also a functional UBD and plays critical roles in NF-κB activation [10]. Remarkably, while neither the UBAN nor the ZF domains shows any preference for K63-linked polyubiquitin chains, they together form a bipartite high-affinity K63-specific UBD [11]. Further structural studies are required to reveal the molecular basis for this specificity.

Recognition of Lys63-linked diubiquitin by UIMs of RAP80

Receptor-associated protein 80 (RAP80) plays a key role in recruiting the Abraxas-BRCC36-BRCA1-BARD1 complex to DNA damage foci for DNA repair by possessing an Abraxas-interacting region. It also contains tandem UIMs, which are single-helical motifs, for interaction with Lys63-linked polyubiquitin. The crystal structure of the tandem UIMs of RAP80 in complex Lys63-linked diubiquitin revealed that the two UIMs of RAP80 and the inter-UIM region form a continuous helix [12,13]. The proximal and the distal ubiquitins of the Lys63-linked diubiquitin interact with UIM1 and UIM2, respectively, in a manner that involves the Ile44 patches (Figure 1D). The specific spacing between the UIMs appears to be critical in the specificity of the interaction for Lys63 linkage, although no direct interaction is present between RAP80 and the Lys63-isopeptide bond [12,13].

Concerted polyubiquitin interactions by ubiquitin receptors in the proteasome

Lys48-linked polyubiquitin recognitions by the two ubiquitin receptors in the proteasome, S5a and Rpn13, have been revealed by elegant crystallographic and NMR studies [14,15]. S5a contains two independent UIMs and its UIM2 preferentially interacts with the proximal ubiquitin in a Lys48-linked diubiquitin (Figure 1E). Rpn13 contains a pleckstrin-like receptor for ubiquitin (Pru) domain and uses loops rather than secondary structural elements for ubiquitin interaction [14]. Dibuqiutin recognition by Rpn13 is mediated mostly by binding to the proximal ubiquitin at the Ile44 patch and to the Lys48 linkage between the proximal and the distal ubiquitin (Figure 1F). It is proposed that in the intact proteasome, Rpn13 and S5a interact simultaneously with tetraubiquitin or a longer polyubiquitin to accomplish polyubiquitin targeting to the proteasome [15].

Specificity for different linkages in DUBs

AMSH-like protease (AMSH-LP) is a Jab1/MPN domain-containing DUB with a Zn-coordinating catalytic core. The crystal structure of Lys63-linked diubiquitin in complex with AMSH-LP revealed that the distal ubiquitin uses the Ile44 hydrophobic patch for interaction with AMSH-LP while the proximal ubiquitin uses an alternative surface [16]. Most strikingly, the surface of AMSH-LP forms a catalytic groove that accommodates the Lys63 side chain of the proximal ubiquitin and the isopeptide-linked C-terminal tail of the distal ubiquitin (Figure 1G). Only the Lys63-linked proximal ubiquitin can bind in the proper orientation for catalysis. Sequence similarity also suggests that the specificity of AMSH for Lys63-linked chains is derived primarily from the interactions with the proximal ubiquitin. Bidentate recognition of both proximal and distal ubiquitins has also been shown as the mechanism of specificity for K48 linkage in the ovarian tumor domain-containing DUB, Otubain 1 [17].

Specific recognition of Lys63-linked diubiquitin conformation by TAB2 and TAB3

Activation of the kinase TAK1 in JNK and NF-κB signaling pathways requires interaction with Lys63-linked polyubiquitin chains by the TAK1-binding proteins, TAB2 and TAB3. TAB2 and TAB3 recognize Lys63-linked chains, but not linear chains, using their C-terminal Np14 zinc finger (NZF) domains. Crystal structures of the TAB2 NZF domain in complex with Lys63-linked diubiquitin or triubiquitin and the TAB3 NZF domain in complex with Lys63-linked diubiquitin reveal that each NZF domain interacts with a diubiquitin using two distinct binding sites and that multiple NZF domains could recognize longer polyubiquitin chains successively [18,19] (Figure 1H). The NZF domains recognize the Ile44 hydrophobic patches of both the proximal and the distal ubiquitins in a diubiquitin. Interestingly, the Lys63-linked isopeptide bond is not involved in the interaction. In contrast, the specificity of the interaction for Lys63-linked chains versus linear chains is conformational as the Met1 residue of the proximal ubiquitin is too far away from the C-terminus of the distal ubiquitin in the diubiquitin complexes with the NZF of TAB2 and TAB3.

Recognition of Lys63-linked diubiquitin by a specific antibody

Interestingly, in an effort to reveal the role of polyubiquitin chains of specific linkages in diverse biological processes, antibodies were developed to recognize Lys63 versus Lys48 linkages [20]. The crystal structure of a Fab fragment of a specific antibody in complex with Lys63-linked diubiquitin revealed that the Fab interacts with the diubiquitin at either side of the isopeptide linkage with limited involvement of the Ile44 patch of either ubiquitin (Figure 1I). It appears that the Fab specifically recognizes a conformation of the diubiquitin that is compatible with the Lys63, but not the Lys48, linkage.