Ubiquitin-conjugating Enzyme Cdc34 and Ubiquitin Ligase Skp1-Cullin-F-box Ligase (SCF) Interact through Multiple Conformations

Daniella Sandoval,Spencer Hill,Amy Ziemba,Steven Lewis,Brian Kuhlman,Gary Kleiger

doi:10.1074/jbc.m114.615559

Abstract

In the ubiquitin-proteasome system, protein substrates are degraded via covalent modification by a polyubiquitin chain. The polyubiquitin chain must be assembled rapidly in cells, because a chain of at least four ubiquitins is required to signal for degradation, and chain-editing enzymes in the cell may cleave premature polyubiquitin chains before achieving this critical length. The ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase SCF are capable of building polyubiquitin chains onto protein substrates both rapidly and processively; this may be explained at least in part by the atypically fast rate of Cdc34 and SCF association. This rapid association has been attributed to electrostatic interactions between the acidic C-terminal tail of Cdc34 and a feature on SCF called the basic canyon. However, the structural aspects of the Cdc34-SCF interaction and how they permit rapid complex formation remain elusive. Here, we use protein cross-linking to demonstrate that the Cdc34-SCF interaction occurs in multiple conformations, where several residues from the Cdc34 acidic tail are capable of contacting a broad region of the SCF basic canyon. Similar patterns of cross-linking are also observed between Cdc34 and the Cul1 paralog Cul2, implicating the same mechanism for the Cdc34-SCF interaction in other members of the cullin-RING ubiquitin ligases. We discuss how these results can explain the rapid association of Cdc34 and SCF.

Highlights

The ubiquitin-conjugating enzyme Cdc34 rapidly associates with ubiquitin ligase SCF to achieve processive ubiquitination
4) Cross-linking occurred between each of the Cdc34 2C proteins and the Cul2 basic canyon (BC) with similar levels of product formation, suggesting that the Cdc34 acidic tail (AT) may interact with the BCs of the cullin-RING ubiquitin ligase (CRL) through a conserved mechanism involving multiple conformations
We propose the following model to explain how interaction of the Cdc34 AT with the Cul1 BC enables the rapid association of Cdc34 with SCF

Summary

Background

The ubiquitin-conjugating enzyme Cdc rapidly associates with ubiquitin ligase SCF to achieve processive ubiquitination. The ubiquitin-conjugating enzyme Cdc and ubiquitin ligase SCF are capable of building polyubiquitin chains onto protein substrates both rapidly and processively; this may be explained at least in part by the atypically fast rate of Cdc and SCF association This rapid association has been attributed to electrostatic interactions between the acidic C-terminal tail of Cdc and a feature on SCF called the basic canyon. The acidic tail promotes the binding of Cdc to SCF through its direct interaction with a conserved basic region on the Cul subunit called the basic canyon This electrostatic interaction enables extremely fast association rates between Cdc and SCF and at least partially explains why ubiquitin chains are processively assembled onto SCF-bound substrates [8]. Our results are most consistent with a model whereby the Cdc acidic tail binds to Cul in multiple conformations, which explains how the tail promotes the rapid association of Cdc with SCF

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION