Structure of a Conserved Dimerization Domain within the F-box Protein Fbxo7 and the PI31 Proteasome Inhibitor

Rebecca Kirk,Heike Laman,Phillip P Knowles,Judith Murray-Rust,Mikhail Lomonosov,El Kahina Meziane,Neil Q Mcdonald

doi:10.1074/jbc.m709900200

Abstract

F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCF(Fbxo7) E3 ligase, and the proteasome inhibitor PI31. SCF(Fbxo7) is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7.Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel alpha/beta-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7.Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCF(Fbxo7). We present a model for FP domain-mediated dimerization of SCF(Fbxo7) and PI31.

Highlights

hepatoma-up-regulated protein (HURP) and cIAP1 have both been reported as substrates for SCFFbxo7-mediated ubiquitination leading to proteasome-mediated degradation [17, 18]
Mapping Protein Interaction Sites within Fbxo7 and Identification of PI31 as an Fbxo7 Binding Partner—To map regions within Fbxo7 responsible for mediating its in vivo interactions with known protein partners, we prepared several deletion constructs based on predicted domain boundaries of Fbxo7 and incorporated an N-terminal T7 epitope
To understand how F-box proteins engage Skp1-Cullin1-F-box protein (SCF) substrates and other potential regulatory proteins through protein-protein interaction, we focused our analysis on the Fbxo7 subunit of the SCFFbxo7

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—A bicistronic expression vector was constructed to prepare recombinant GSTtagged Fbxo71⁄7Skp complexes for affinity purification. To obtain the native PI31 FP domain structure free of introduced mutations, a partial model was built into this map and was subsequently positioned by molecular replacement into the native unit cell (dataset 1) Refinement against this native dataset collected in house produced the final model (Table 1, dataset 1). Analytical Ultracentrifugation—For analytical ultracentrifugation (AUC) experiments, recombinant proteins were stored and analyzed in a buffer containing 20 mM Tris-HCl, pH 8, 50 mM NaCl, and 5 mM ␤-mercaptoethanol. Cells were lysed in hypotonic lysis buffer (10 mM Tris-HCL, pH 7.5, 10 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) and protease inhibitor mixture for 10 min on ice before the addition of NaCl to a final concentration of 150 mM. Anti-rabbit IgG and anti-mouse IgG antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. and Jackson ImmunoResearch Laboratories

RESULTS

97 Cullin1

DISCUSSION