Structural Basis of Dimerization-dependent Ubiquitination by the SCFFbx4 Ubiquitin Ligase

Yunfeng Li,Bing Hao

doi:10.1074/jbc.m110.111518

Abstract

The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F- box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF(Fbx4) (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, whereas the C-terminal domain of the protein adopts a compact alpha/beta fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.

Highlights

SCF,2 the prototype of the cullin-RING ligases, is built in a modular format that is conserved from yeast to humans [6, 8]
The accumulated evidence suggests that the large number and rich diversity of F-box proteins allow the recruitment of numerous, diverse substrates and position them optimally for the ubiquitination reaction, an important feature of the SCF E3s [13, 14]
The ubiquitin-protein ligase SCFFbx4 was recently identified as the E3 responsible for the ubiquitination and subsequent degradation of the cell cycle regulator cyclin D1 and telomeric DNA-binding protein Pin2 [25, 26]

Summary

Introduction

SCF, the prototype of the cullin-RING ligases, is built in a modular format that is conserved from yeast to humans [6, 8]. Biophysical, and biochemical studies show that: 1) the full-length Fbx is monomeric and essentially inactive in ubiquitinating Pin in vitro; 2) a loop connecting the linker domain to the C-terminal substrate-binding domain of Fbx is crucial for dimerization, and the head-to-tail dimerization configuration of a truncated Fbx protein likely plays a role in promoting substrate binding and ubiquitin transfer; 3) the extreme N-terminal region of Fbx is important for interaction with Cul1-Rbx and may be responsible for regulating Fbx dimerization and ubiquitination activity; and 4) Fbx possesses a previously uncharacterized type of substrate recognition domain. Our results delineate a critical role for Fbx dimerization in regulating SCFFbx ubiquitination activity and support the notion that inactivation of the Fbx ligase in human cancer may result from mutations that impair ligase dimerization

Results

Discussion

Conclusion