Dataset Information

A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases.

ABSTRACT: How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s.

SUBMITTER: Calabrese MF

PROVIDER: S-EPMC3245743 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases.

Calabrese Matthew F MF Scott Daniel C DC Duda David M DM Grace Christy R R CR Kurinov Igor I Kriwacki Richard W RW Schulman Brenda A BA

Nature structural & molecular biology 20110717 8

How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s. ...[more]

PMID: 21765416

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Project description:UnlabelledThe rotavirus nonstructural protein NSP1 acts as an antagonist of the host antiviral response by inducing degradation of key proteins required to activate interferon (IFN) production. Protein degradation induced by NSP1 is dependent on the proteasome, and the presence of a RING domain near the N terminus has led to the hypothesis that NSP1 is an E3 ubiquitin ligase. To examine this hypothesis, pulldown assays were performed, followed by mass spectrometry to identify components of the host ubiquitination machinery that associate with NSP1. Multiple components of cullin RING ligases (CRLs), which are essential multisubunit ubiquitination complexes, were identified in association with NSP1. The mass spectrometry was validated in both transfected and infected cells to show that the NSP1 proteins from different strains of rotavirus associated with key components of CRL complexes, most notably the cullin scaffolding proteins Cul3 and Cul1. In vitro binding assays using purified proteins confirmed that NSP1 specifically interacted with Cul3 and that the N-terminal region of Cul3 was responsible for binding to NSP1. To test if NSP1 used CRL3 to induce degradation of the target protein IRF3 or β-TrCP, Cul3 levels were knocked down using a small interfering RNA (siRNA) approach. Unexpectedly, loss of Cul3 did not rescue IRF3 or β-TrCP from degradation in infected cells. The results indicate that, rather than actively using CRL complexes to induce degradation of target proteins required for IFN production, NSP1 may use cullin-containing complexes to prevent another cellular activity.ImportanceThe ubiquitin-proteasome pathway plays an important regulatory role in numerous cellular functions, and many viruses have evolved mechanisms to exploit or manipulate this pathway to enhance replication and spread. Rotavirus, a major cause of severe gastroenteritis in young children that causes approximately 420,000 deaths worldwide each year, utilizes the ubiquitin-proteasome system to subvert the host innate immune response by inducing the degradation of key components required for the production of interferon (IFN). Here, we show that NSP1 proteins from different rotavirus strains associate with the scaffolding proteins Cul1 and Cul3 of CRL ubiquitin ligase complexes. Nonetheless, knockdown of Cul1 and Cul3 suggests that NSP1 induces the degradation of some target proteins independently of its association with CRL complexes, stressing a need to further investigate the mechanistic details of how NSP1 subverts the host IFN response.

Dataset Information

A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases.

Publications

A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases.

Similar Datasets

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