Structure and dynamics of the ASB9 CUL-RING E3 Ligase

Ryan J Lumpkin,Richard W Baker,Andres E Leschziner,Elizabeth A Komives

doi:10.1038/s41467-020-16499-9

Ryan J Lumpkin, Richard W Baker + Show 2 more

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https://doi.org/10.1038/s41467-020-16499-9

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Abstract

The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. Here we report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. To date, no full structures are available either for a substrate-bound ASB nor for CUL5. Hydrogen–deuterium exchange (HDX-MS) mapped onto a full structural model of the ligase revealed long-range allostery extending from the substrate through CUL5. We propose a revised allosteric mechanism for how CUL-E3 ligases function. ASB9 and CUL5 behave as rigid rods, connected through a hinge provided by ELOB/C transmitting long-range allosteric crosstalk from the substrate through CUL5 to the RBX2 flexible linker.

Highlights

The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different Suppressor of Cytokine Signaling (SOCS)-box-containing substrate receptors, determining the substrate specificity of the ligase
To study the assembly of the full CUL5 complex, we prepared a number of different sub-complexes of the E3 ligase complex containing Creatine Kinase brain-type (CKB)-ASB9-Elongin B (ELOB)/C-CUL5-RBX2 (Supplementary Fig. 1)
Initial experiments preparing sub-complexes revealed that we could prepare CKB-ASB9ELOB/C and mix that with ELOB/C-CUL5-RBX2 to form the full ligase containing only one ELOB/C subunit. These experiments revealed that ELOB/C could be exchanged whereas the CKB-ASB9 interaction and the CUL5-RBX2 interaction were essentially irreversible

Summary

Introduction

The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. We report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/ C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. CRLs share a common structure, composed of a substrate receptor(s), a CUL, and a RING-box (RBX) protein. The 18 ASB proteins comprise the largest family of SOCS-box domain-containing E3 ligase substrate receptors. Each ASB protein contains an ankyrin repeat domain and a SOCS-box domain with CUL5-box and BC box motifs, and they associate with CUL5 through the Elongin B and C (ELOB/C) adapter proteins[4,5,6]. While some details have been shown in a fragmented manner, a full mechanistic understanding of the ASB9 CRL assembly, substrate recognition, and dynamics remain unknown

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