The molecular determinants for distinguishing between ubiquitin and NEDD8 by USP2

Shin, Yung-Cheng; Chen, Jou-Han; Chang, Shih‐Chung

doi:10.1038/s41598-017-02322-x

Cited by 11 publications

(12 citation statements)

References 47 publications

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“…[67][68][69] However, due to Arg72 in ubiquitin, and Ala72 in NEDD8, some UBDs can discriminate between ubiquitin and NEDD8. 70,71 The stronger cationπ electron interaction between Arg and Trp compared to Arg and Phe, 72,73 further supports our suggestion of selectivity in binding between CSNAP and DSS1. In DSS1, acidic and hydrophobic residues interact with the hydrophobic patch on ubiquitin formed by Ile13, Ile44, and Leu69, flanked by two basic regions.…”

Section: Discussionsupporting

confidence: 79%

The disordered PCI‐binding human proteins CSNAP and DSS1 have diverged in structure and function

et al. 2021

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Intrinsically disordered proteins (IDPs) regularly constitute components of larger protein assemblies contributing to architectural stability. Two small, highly acidic IDPs have been linked to the so-called PCI complexes carrying PCI-domain subunits, including the proteasome lid and the COP9 signalosome. These two IDPs, DSS1 and CSNAP, have been proposed to have similar structural propensities and functions, but they display differences in their interactions and interactome sizes. Here we characterized the structural properties of human DSS1 and CSNAP at the residue level using NMR spectroscopy and probed their propensities to bind ubiquitin. We find that distinct structural features present in DSS1 are completely absent in CSNAP, and vice versa, with lack of relevant ubiquitin binding to CSNAP, suggesting the two proteins to have diverged in both structure and function. Our work additionally highlights that different local features of seemingly similar IDPs, even subtle sequence variance, may endow them with different functional traits. Such traits may underlie their potential to engage in multiple interactions thereby impacting their interactome sizes.

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Section: Discussionsupporting

confidence: 79%

The disordered PCI‐binding human proteins CSNAP and DSS1 have diverged in structure and function

et al. 2021

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“…In summary, we suggest that the exposed side chains in this region represent a peculiar distribution of hindered (threonines), aromatic (phenylalanine), polar charges (lysine and glutamic acid), and flexibility sources (glycine), which are able to strongly influence, in different ways, the protein recognition with a determined substrate. Intriguingly, residues 4, 12, 14, and 64 were recently shown to be molecular determinants for distinguishing between ubiquitin and NEDD8 by ubiquitin-specific peptidase 2 (USP2) [29], thus confirming their key role in the discrimination mechanism.…”

Section: Discussionmentioning

confidence: 96%

CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

et al. 2019

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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.

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“…In summary, we can suggest that the exposed side chains in this region represent a peculiar distribution of hindered (threonines), aromatic (phenylalanine), polar charges (lysine and glutamic acid) and flexibility sources (glycine) which are able to strongly influence, in different ways, the protein recognition with a determined substrate. Intriguingly, residues 4, 12, 14 and 64 have been recently shown to be molecular determinants for distinguishing between ubiquitin and NEDD8 by Ubiquitin-specific peptidase 2 (USP2) [32], thus confirming their key role in the discrimination mechanism.…”

Section: Discussionmentioning

confidence: 87%

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

Nepravishta¹,

Ferrentino²,

Mandaliti³

et al. 2019

Preprint

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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 (Leu-8, Ile-44, Val-70). Nevertheless, 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 recognizes ubiquitin. Here, we describe the structure of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis and NMR spectroscopy of the 15N isotopically labelled 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 CUE (Coupling of ubiquitin conjugation to ER degradation) domain family, where an invariant proline, usually following a phenylalanine, is required for binding to ubiquitin. Interestingly, the UBD of N4BP1 is evolutionary related to CUBAN (Cullin binding domain associating with NEDD8) (40% identity and 47% similarity), a protein module that also recognizes the ubiquitin-like NEDD8, which is the closest relative of ubiquitin (58% identity and 80% similarity). By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the UBD of N4BP1 lacks the NEDD8 binding properties observed in CUBAN and it recognizes the Ile44-centered patch of ubiquitin through a dedicated binding site, which share some of the features observed in the CUE domain family. Moreover, we show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both the CUBAN and the UBD of N4BP1 are poly-ubiquitinated in cells. This modification is dependent on the presence of a functional domain. We believe that the structural and functional characterization of this novel UBD will allow a deeper understanding of the molecular mechanisms governing N4BP1 function, while at the same time providing a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

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The molecular determinants for distinguishing between ubiquitin and NEDD8 by USP2

Cited by 11 publications

References 47 publications

The disordered PCI‐binding human proteins CSNAP and DSS1 have diverged in structure and function

The disordered PCI‐binding human proteins CSNAP and DSS1 have diverged in structure and function

CoCUN, a Novel Ubiquitin Binding Domain Identified in N4BP1

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

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