Dataset Information

CUBAN, a Case Study of Selective Binding: Structural Details of the Discrimination between Ubiquitin and NEDD8.

ABSTRACT: The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The 15N HSQC NMR perturbation pattern of the labeled CUBAN domain, when combined with either NEDD8 or ubiquitin, shows a clear involvement of hydrophobic residues that characterize the early stages of these interactions. After a slow conformational selection step, hydrophobic and then neutral and polar interactions take place, which drive the correct orientation of the CUBAN domain, leading to differences in the recognition scheme of NEDD8 and ubiquitin. As a result, a cascade of induced fit steps seems to determine the structural preference shown for NEDD8 and therefore the basis of the selectivity of the CUBAN domain. Finally, molecular dynamics analysis was performed to determine by fluctuations the internal flexibility of the CUBAN/NEDD8 complex. We consider that our results, based on a structural investigation mainly focused on the early stages of the recognition, provide a fruitful opportunity to report the different behavior of the same protein with two highly similar binding partners.

SUBMITTER: Santonico E

PROVIDER: S-EPMC6429362 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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CUBAN, a Case Study of Selective Binding: Structural Details of the Discrimination between Ubiquitin and NEDD8.

Santonico Elena E Nepravishta Ridvan R Mandaliti Walter W Castagnoli Luisa L Cesareni Gianni G Paci Maurizio M

International journal of molecular sciences 20190308 5

The newly identified CUBAN (Cullin binding domain associating with NEDD8) domain recognizes both ubiquitin and the ubiquitin-like NEDD8. Despite the high similarity between the two molecules, CUBAN shows a clear preference for NEDD8, free and conjugated to cullins. We previously characterized the domain structure, both alone and in complex with NEDD8. The results here reported are addressed to investigate the determinants that drive the selective binding of CUBAN towards NEDD8 and ubiquitin. The ...[more]

PMID: 30857167

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Project description:BACKGROUND:Neuroinflammation, typified by elevated levels of interleukin-1 (IL-1) ? and ?, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1? on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8. METHODS:Immunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1? effects on PINK1, P-Ub, parkin, P-parkin, and GSK3?-as well as phosphorylation of parkin by GSK3?-were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3?. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1?, IL-1?, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates. RESULTS:IL-1?, IL-1?, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1?-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1?-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3?-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser65 for parkin activation. CONCLUSIONS:The promotion of parkin- and NEDD8-mediated ubiquitination by IL-1? is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1? suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1? elevation, easily envisioned considering its early induction in Down's syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.

Project description:Post-translational modifications by ubiquitin and ubiquitin-like proteins (Ubls) have known roles in a myriad of cellular processes. Ubiquitin- and Ubl-binding domains transmit the information conferred by these post-translational modifications by recognizing functional surfaces and, when present, different chain structures. Numerous domains binding to ubiquitin have been characterized and their structures solved. Analogously, motifs selectively interacting with SUMO (small ubiquitin-like modifier) have been identified in several proteins and their role in SUMO-dependent processes investigated. On the other hand, proteins that specifically recognize other Ubl modifications are known only in a few cases. The high sequence identity between NEDD8 and ubiquitin has made the identification of specific NEDD8-binding domains further complicated due to the promiscuity in the recognition by several ubiquitin-binding domains. Two evolutionarily related domains, called CUBAN (cullin-binding domain associating with NEDD8) and CoCUN (cousin of CUBAN), have been recently described. The CUBAN binds monomeric NEDD8 and neddylated cullins, but it also interacts with di-ubiquitin chains. Conversely, the CoCUN domain only binds ubiquitin. CUBAN and CoCUN provide an intriguing example of how nature solved the issue of promiscuity versus selectivity in the recognition of these two highly related molecules. The structural information available to date suggests that the ancestor of CUBAN and CoCUN was a three-helix bundle domain that diversified in KHNYN (KH and NYN domain-containing) and N4BP1 (NEDD4-binding protein-1) by acquiring different features. Indeed, these domains diverged towards two recognition modes, that recall respectively the electrostatic interaction utilized by the E3-ligase RBX1/2 in the interaction with NEDD8, and the hydrophobic features described in the recognition of ubiquitin by CUE (coupling ubiquitin conjugation to ER degradation) domains. Intriguingly, CUBAN and CoCUN domains are only found in KHNYN and N4BP1, respectively, both proteins belonging to the PRORP family whose members are characterized by the combination of protein modules involved in RNA metabolism with domains mediating ubiquitin/NEDD8 recognition. This review recapitulates the current knowledge and recent findings of CUBAN and CoCUN domains and the proteins containing them.

Dataset Information

CUBAN, a Case Study of Selective Binding: Structural Details of the Discrimination between Ubiquitin and NEDD8.

Publications

CUBAN, a Case Study of Selective Binding: Structural Details of the Discrimination between Ubiquitin and NEDD8.

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