Selectivity of the CUBAN domain in the recognition of ubiquitin and NEDD8

Abstract

Among the members of the ubiquitin‐like (Ubl) protein family, neural precursor cell expressed developmentally down‐regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here, we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin‐binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl end of the protein KHNYN, which has a stark preference for NEDD8. Given its ability to bind neddylated cullins, we have named this domain CUBAN (Cullin‐Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill‐defined protein, whose function is hitherto only predicted.

The Ubiquitin <em>versus</em> NEDD8 Binding Preference of NEDD4 Binding Protein 1 (N4BP1) Is Based on a Mutual Conformational Perturbation

“…By panning a human brain phage-displayed cDNA library using ubiquitin as bait, we identified a shorter fragment of N4BP1 (aa 813-896) that shows ubiquitin binding features and we further reduced the functional region to the last 50 amino acids of human N4BP1 (847-896aa) by means of biochemical approaches. Based on mutational analysis, we have shown that the interaction between N4BP1 and ubiquitin involves the Ile44-hydrophobic patch [11], in analogy with what has been observed in the majority of the UBDs characterized so far.…”

“…Interestingly, the minimal ubiquitin-binding region in N4BP1, including residues 849-896, shares 40% identity with the evolutionary related CUBAN domain, recently identified in the evolutionary related KHNYN protein [11]. The CUBAN has been shown to bind NEDD8 with an affinity that is higher than the value measured for monomeric ubiquitin [11]. In addition to interacting with neddylated cullins thanks to the direct recognition of the NEDD8 molecule, the CUBAN domain binds ubiquitin chains and polyubiquitinated proteins.…”

“…Fenner and collaborators recently identified N4BP1 as a potential polyubiquitin chains binding protein. A previous analysis, performed with the phage display approach, enabled us to identify the region spanning residues 813-896 in N4BP1 as the one showing ubiquitin binding features ( Figure 1A) [11]. The bioinformatics analysis of this region, performed with Simple Modular Architecture Research Tool (SMART) database, reported the presence of a CUE domain whose primary sequence, however, is too divergent from known CUE domain sequences to be reliably considered a member of this group ( Figure 1B) [11].…”

“…In addition to interacting with neddylated cullins thanks to the direct recognition of the NEDD8 molecule, the CUBAN domain binds ubiquitin chains and polyubiquitinated proteins. This dual capability is due to the presence of two distinct binding sites that are in close proximity, as evidenced by the observation that the interaction of the CUBAN domain with ubiquitin chains competes out NEDD8 binding [11].…”

“…Nevertheless, the structural analysis shows that key differences in the ubiquitin-binding domain of N4BP1 do not support the inclusion of this novel domain neither in the CUE nor in the UBA domain families. Conversely, the UBD of N4BP1 shares common features with the Cullin binding domain associating with NEDD8 (CUBAN), recently identified in the evolutionary related KHNYN protein [11] (Santonico et al, 2019, submitted). The observed similarity, both at the primary sequence and structural level, supports the identification of a novel protein domain family having the CUBAN domain and the UBD of N4BP1 as unique members.…”