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The molecular determinants of NEDD8 specific recognition by human SENP8.

ABSTRACT: Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. This CrUb-CrNEDD8 protein was termed CrRUB1 (related to ubiquitin 1) by analogy with a similar protein in Arabidopsis thaliana (AtRUB1). Since there is high sequence identity in comparison to the corresponding human proteins (97% for ubiquitin and 84% for NEDD8), a His-CrRUB1-glutathione S-transferase (GST) fusion construct was adopted as the alternative substrate to characterize the specificity of NEDD8-specific peptidase SENP8 for CrNEDD8. The data showed that SENP8 only cleaved the peptide bond beyond the di-glycine motif of CrNEDD8 and His-RUB1 was subsequently generated, confirming that SENP8 has exquisite specificity for CrNEDD8 but not CrUb. To further determine the basis of this specificity, site-directed mutagenesis at earlier reported putative molecular determinants of NEDD8 specific recognition by SENP8 was performed. We found that a single N51E mutation of CrNEDD8 completely inhibited its hydrolysis by SENP8. Conversely, a single E51N mutation of CrUb enabled this ubiquitin mutant to undergo hydrolysis by SENP8, revealing that a single residue difference at the position 51 contributes substantially to the substrate selectivity of SENP8. Moreover, the E51N/R72A double mutant of the CrUb subdomain can further increase the efficiency of cleavage by SENP8, indicating that the residue at position 72 is also important in substrate recognition. The E51N or R72A mutation of CrUb also inhibited the hydrolysis of CrUb by ubiquitin-specific peptidase USP2. However, USP2 cannot cleave the N51E/A72R double mutant of the CrNEDD8 subdomain, suggesting that USP2 requires additional recognition sites.

SUBMITTER: Shin YC

PROVIDER: S-EPMC3215745 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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The molecular determinants of NEDD8 specific recognition by human SENP8.

Shin Yung-Cheng YC Tang Siao-Jing SJ Chen Jou-Han JH Liao Pei-Han PH Chang Shih-Chung SC

PloS one 20111114 11

Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. ...[more]

PMID: 22110750

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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.

Project description:Protein kinases are key enzymes in many signal transduction pathways, and play a crucial role in cellular proliferation, differentiation, and various cell regulatory processes. However, aberrant function of kinases has been associated with cancers and many other diseases. Consequently, competitive inhibition of the ATP binding site of protein kinases has emerged as an effective means of curing these diseases. Over the past three decades, thousands of protein kinase inhibitors (PKIs) with varying molecular frames have been developed. Large-scale data mining of the Protein Data Bank resulted in a database of 2139 non-redundant high-resolution X-ray crystal structures of PKIs bound to protein kinases. This provided us with a unique opportunity to study molecular determinants for the molecular recognition of PKIs. A chemoinformatic analysis of 2139 PKIs resulted in findings that PKIs are "flat" molecules with high aromatic ring counts and low fractions of sp3 carbon. All but one PKI possessed one or more aromatic rings. More importantly, it was found that the average weighted hydrogen bond count is inversely proportional to the number of aromatic rings. Based on this linear relationship, we put forward the exchange rule of hydrogen bonding interactions and non-bonded π-interactions. Specifically, a loss of binding affinity caused by a decrease in hydrogen bonding interactions is compensated by a gain in binding affinity acquired by an increase in aromatic ring-originated non-bonded interactions (i.e., π-π stacking interactions, CH-π interactions, cation-π interactions, etc.), and vice versa. The very existence of this inverse relationship strongly suggests that both hydrogen bonding and aromatic ring-originated non-bonded interactions are responsible for the molecular recognition of PKIs. As an illustration, two representative PKI-kinase complexes were employed to examine the relative importance of different modes of non-bonded interactions for the molecular recognition of PKIs. For this purpose, two FDA-approved PKI drugs, ibrutinib and lenvatinib, were chosen. The binding pockets of both PKIs were thoroughly examined to identify all non-bonded intermolecular interactions. Subsequently, the strengths of interaction energies between ibrutinib and its interacting residues in tyrosine kinase BTK were quantified by means of the double hybrid DFT method B2PLYP. The resulting energetics for the binding of ibrutinib in tyrosine kinase BTK showed that CH-π interactions and π-π stacking interactions between aromatic rings of the drug and hydrophobic residues in its binding pocket dominate the binding interactions. Thus, this work establishes that, in addition to hydrogen bonding, aromatic rings function as important molecular determinants for the molecular recognition of PKIs. In conclusion, our findings support the following pharmacophore model for ATP-competitive kinase inhibitors: a small molecule features a scaffold of one or more aromatic rings which is linked with one or more hydrophilic functional groups. The former has the structural role of acting as a scaffold and the functional role of participating in aromatic ring-originated non-bonded interactions with multiple hydrophobic regions in the ATP binding pocket of kinases. The latter ensure water solubility and form hydrogen bonds with the hinge region and other hydrophilic residues of the ATP binding pocket.

Dataset Information

The molecular determinants of NEDD8 specific recognition by human SENP8.

Publications

The molecular determinants of NEDD8 specific recognition by human SENP8.

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