CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

Abstract

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

Old and New Concepts in Ubiquitin and NEDD8 Recognition

“…An FW motif ( 646 Phe-Trp 647 ), corresponding to the FP sequence characterized in CoCUN and also here positioned inside the extended loop1, is not involved in the recognition of NEDD8 nor in the ubiquitination of CUBAN. On the contrary, this amino acid couple points towards the core of the domain, and it appears to reduce the flexibility of loop1, thus performing a stabilizing function (Figure 3) [64,99]. From a structural point of view, the marked difference observed in the FP and FW motifs is the result of the different spatial distribution of the helix-2 in CUBAN compared to other three-helix bundle domains, as previously discussed (Figure 2).…”

“…Indeed, similarly to CUE/ubiquitin complexes, the FP motif recognizes the canonical hydrophobic patch of ubiquitin, and the Pro/Ala mutation disrupts the interaction between the two partners. In addition, the wild-type domain, but not the PA mutant, is ubiquitinated when transiently transfected in cells [99]. This feature is shared among CUE, UIM, MIU and A20 ZnF-containing proteins in which the ubiquitin-binding properties are coupled with the ubiquitination of the protein in which they are found, a process called coupled-monoubiquitination [17,106,107].…”

“…The interaction of CUBAN with neddylated cullins is independent from ubiquitination and only relies on the presence of the conjugated Ubl [64]. The CoCUN (cousin of CUBAN) domain, found in N4BP1 and spanning the C-terminal residues 847-896, shares 40% identity and 47% similarity with CUBAN [99]. The measured binding affinity of CoCUN for monomeric ubiquitin is 50 µM and, differently from the related CUBAN, it does not show any NEDD8-binding property.…”

“…As previously discussed, an important challenge in the field of ubiquitin-like signaling is to unravel the mechanisms underlying the specificity of ubiquitin-binding modules for Ubl molecules. The phage display approach has been recently applied to understand the determinants of ubiquitin and NEDD8 specificity recognition, leading to the identification of two novel ubiquitin and NEDD8 binding domains in the two evolutionary related proteins KHNYN (KH and NYN domain-containing) and N4BP1 (NEDD4-binding protein-1) [64,98,99]. Compared to previously characterized ubiquitin-binding domains, the selected regions show unique features.…”

“…From a structural point of view, the investigation by NMR spectroscopy of CUBAN and CoCUN domains reported the presence of structural elements that are common to other UBDs belonging to the most populated category represented by a three-bundle helix, such as UBA and CUE [16,99]. Compared to the latter, a feature that is common to CUBAN and CoCUN domains is the unusually extended loop1, which is also typified by pronounced flexibility.…”