Structure of a Conserved Dimerization Domain within the F-box

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

Received for publication, December 4, 2007, and in revised form, May 12, 2008 Published, JBC Papers in Press, May 20, 2008, DOI 10.1074/jbc.M709900200

Rebecca Kirk‡1, Heike Laman§2, Phillip P. Knowles‡, Judith Murray-Rust‡, Mikhail Lomonosov§3,El Kahina Meziane§4, and Neil Q. McDonald‡¶5

From the ‡Structural Biology Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, LondonWC2A 3PX, United Kingdom, §Department of Pathology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QP, UnitedKingdom, and ¶School of Crystallography, Birkbeck College, Malet St., London WC1E 7HX, United Kingdom

F-box proteins are the substrate-recognition componentsof the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases.Here we report a structural relationship between Fbxo7, acomponent of the SCFFbxo7 E3 ligase, and the proteasomeinhibitor PI31. SCFFbxo7 is known to catalyze the ubiquitina-tion of hepatoma-up-regulated protein (HURP) and theinhibitor of apoptosis (IAP) protein but also functions as anactivator of cyclin D-Cdk6 complexes. We identify PI31 as anFbxo7�Skp1 binding partner and show that this interactionrequires anN-terminal domain present in both proteins that weterm the FP (Fbxo7/PI31) domain. The crystal structure of thePI31 FP domain reveals a novel�/�-fold. Biophysical andmuta-tional analyses are used to map regions of the PI31 FP domainmediating homodimerization and required for heterodimeriza-tion with Fbxo7�Skp1. Equivalent mutations in Fbxo7 ablateinteraction with PI31 and also block Fbxo7 homodimerization.Knockdown of Fbxo7 does not affect PI31 levels arguing againstPI31 being a substrate for SCFFbxo7. We present a model for FPdomain-mediated dimerization of SCFFbxo7 and PI31.

The levels of many regulatory and misfolded proteins arecontrolled by the ubiquitin-proteasome system (1, 2). A seriesof enzymes termed E1 (ubiquitin-activating enzyme), E2 (ubiq-uitin carrier protein), and E36 (ubiquitin ligase) act in the ubiq-uitin-proteasome system as part of a concerted cascade to acti-vate and conjugate ubiquitin via an isopeptide linkage toprotein substrates (2). Ubiquitin modification can affect theactivity, localization, sorting, and stability of protein substrates

(2–4). Recognition and recruitment of protein substrates to theubiquitin-proteasome system machinery resides within themultisubunit E3 ubiquitin ligases, the largest group of which isthe Skp1-Cullin1-F-box protein (SCF) family (1, 5, 6). Allknown SCF E3 ligases bind an E2 (ubiquitin carrier protein)enzyme through a RING finger-containing subunit (Rbx1) andutilize their F-box subunit to recruit substrates. Although clas-sified by the presence of an F-box (Fbx) motif (7), F-box pro-teins can be further divided into three classes depending onadditional structural elements: Fbxw (WD40 motifs), Fbxl(with leucine-rich repeats), and Fbxo (F-box domain only) (8,9). Recent structural data have shown how the Fbxw proteinCDC4 (Fbw7) uses the center of the WD40 propeller to recog-nize the phosphorylated epitope of its substrate, cyclin E (10,11). Structural data also exist for the Fbxl protein Skp2 (Fbl1) aspart of the SCFSkp2 complex bound to phosphorylated p27 (12).In contrast, little is known about how Fbxo proteins recognizetheir substrates or indeed whether other substrate-targetingdomains exist within this subgroup.Fbxo7 has been characterized recently as a member of the

Fbxo subgroup and is conserved among higher eukaryotes (8,13). As well as an F-box motif (residues 329–375), which inter-acts with Skp1 (8, 9, 13, 14), Fbxo7 has anN-terminal ubiquitin-like (Ubl) domain (residues 1–78) (13, 15) (Fig. 1A), which isthought to mediate interactions with ubiquitin receptor pro-teins bearing ubiquitin binding domains (16). The C terminusof Fbxo7 contains a proline-rich region (PRR) but lacks anypredicted secondary structure. HURP (hepatoma up-regulatedprotein) and cIAP1 (an inhibitor of apoptosis protein) haveboth been reported as substrates for SCFFbxo7-mediated ubiq-uitination leading to proteasome-mediated degradation (17,18). Each binds to Fbxo7 through its C-terminal proline-richregion (17, 18). Fbxo7 also acts as an assembly factor for cyclinD-Cdk6 complexes by virtue of its interaction with D-type cyc-lins and Cdk6 (13).In this study a combination of structural, biochemical, and

genetic approaches were used to identify and characterizeregions of Fbxo7 involved in protein interaction as a means toidentify putative substrates of SCFFbxo7. Unexpectedly weuncovered a structural link between a domain from Fbxo7 anda related domain from PI31, a regulatory subunit of the immu-noproteasome which is an in vitro inhibitor of the 20 S protea-some (19–22). The crystal structure of this domain from PI31revealed a novel �/�-fold and two distinct intermolecular con-tact surfaces. We used this structure to guide the design of

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2VT8) have been deposited inthe Protein Data Bank, Research Collaboratory for Structural Bioinformatics,Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Recipient of a Barbara Mary Hill Fellowship from Cancer Research UK.Current address: Research Institute of Molecular Pathology, 1030 Vienna,Austria.

2 To whom correspondence may be addressed. Tel.: 44-1223-333-722; E-mail:[email protected].

3 Funded by Cancer Research UK.4 Funded by Association for International Cancer Research.5 To whom correspondence may be addressed. Tel.: 44-207-269-3259; E-mail:

[email protected] The abbreviations used are: E3, ubiquitin ligase; HURP, hepatoma up-regu-

lated protein; IAP, inhibitor of apoptosis; ITC, isothermal calorimetry; SCF,Skp1-Cul1-F box protein; Ubl, ubiquitin-like; PRR, proline-rich region; FP,Fbxo7/PI31; GST, glutathione S-transferase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 32, pp. 22325–22335, August 8, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

AUGUST 8, 2008 • VOLUME 283 • NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 22325

by guest on February 19, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

site-specific substitutions to probe surfaces of the FP domainfrom PI31 responsible for homodimerization and for het-erodimeric interaction with Fbxo7. We discuss a possible rolefor FP domain-mediated dimerization for SCFFbxo7 and PI31function.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—A bicistronic expres-sion vector was constructed to prepare recombinant GST-tagged Fbxo7�Skp1 complexes for affinity purification. For allexperiments the Skp1 cDNA encompassed residues 1–146 andlacked the C-terminal H8 helix as previously described (23).Human Fbxo7 (residues 129–398) (Swiss-Prot Q9Y3I1)-Skp1(residues 1–146) (Swiss-Prot P63208), and Fbxo7 (residues169–398)-Skp1 (residues 1–146) constructs were produced asfollows. The FBXO7 gene was cloned into pGEX(KG) (24). TheSKP1 gene was amplified by PCR from a human cDNA libraryusing a 5� primer which contained a ribosome binding siteand then subcloned into BlueScript (Stratagene). The RBS-SKP1 insert was then subcloned into pGEX(KG)-Fbxo7. Forall experiments the Skp1 encompassed residues 1–146(Skp1�H8), which lacks the C-terminal H8 helix aiding expres-sion and solubility (23). Fbxo7(169–398)-Skp1�H8 (V253E)mutant was produced using the QuikChange system (Strat-agene). Residues 1–151 of human PI31 (Swiss-Prot Q92530)were amplified by PCR and cloned into the pET28a expressionplasmid (Novagen). Mutant PI31 constructs (L7M, V6R, andI83E/I90E) and mutant Fbxo7 constructs (V253E and V175E)were produced using the QuikChange system (Stratagene). Allconstructs were verified by DNA sequencing.The expression plasmids described above were transformed

into Escherichia coli strain BL21(DE3), and cultures weregrown in Luria broth at 37 °C to an A600 of 0.6. Recombinantprotein expression was induced by the addition of isopropyl-�-D-1-thiogalactopyranoside to a final concentration of 250 �Mfor 3 h at 37 °C. Bacteria were harvested by centrifugation andresuspended in lysis buffer (100mMNaCl, 50mMTris-HCl, pH8, 10 mM benzimidine, 5 mM �-mercaptoethanol, 1 mM phen-ylmethylsulfonyl fluoride). Proteins were purified from the sol-uble fraction using either glutathione-Sepharose (Fbxo7�Skp1)or nickel-nitrilotriacetic acid affinity beads (PI31) by batchpurification and cleaved from the beads with thrombin at 4 °C.PI31 and Fbxo7�Skp1 proteins were then purified by size exclu-sion chromatography using Superdex 75 and Superdex 200 col-umns, respectively. Columns were equilibrated and run in 20mM Tris-HCl, 50 mM NaCl, pH 8, and 1 mM dithiothreitol.Selenomethionine was incorporated by transforming theexpression plasmid into theE. colimethionine auxotroph strainB834(DE3) grown on minimal medium supplemented by sele-nium methionine using standard procedures. Selenomethi-onine-labeled protein and mutants were prepared using a sim-ilar procedure to the native protein as described above.Crystallization and Data Collection—The PI31 FP domain

was crystallized by vapor diffusion in sitting drops containing 2�l of protein (8 mg/ml in 20 mM Tris-HCl, pH 8.0) and 2 �l ofwell solution (20% polyethylene glycol 3350 and 0.1 M ammo-nium iodide) at room temperature. Crystals grewwithin 1weekto roughly 0.2 mm. Selenomethionine-substituted wild type FP

domain and L7M mutant protein crystallized under the sameconditions as the native protein.Datawere collected for crystalsof human PI31 FP domain, selenomethionine-substituted FPdomain, and the selenomethionine-substituted L7M mutant(Table 1). In each case a single crystal was flash-cooled to 100 Kin a nitrogen gas stream using 20% (v/v) ethylene glycol as acryoprotectant. The CCP4 suite of programs (25) were used fordata processing, andREFMACandCOOTwere used for refine-ment and model building (26, 27).Structure Determination—Initial attempts to solve the struc-

ture using selenomethionine single wavelength anomalous dif-fraction data were unsuccessful due to the centrosymetricarrangement of the two selenium sites from twomethionines innative PI31 FP domain (data not shown). A third methioninesite was engineered by an L7M mutation leading to crystals ofselenomethionyl-labeled protein, and subsequently datasetswere collected at the selenium peak wavelength (Table 1, data-sets 2 and 3). The selenium substructure from dataset 2 wasfound by SHELXD/E and used to calculate SAD phases with acontrast of 0.403 and connectivity of 0.877 (incorrect hand hada contrast of 0.314 and connectivity of 0.845). Phases wereimproved by 2-fold non-crystallographic averaging withindataset 2 (initial FOM 0.45, final FOM 0.64) using DM (28) andcross-crystal averagingwith dataset 3 (Table 1, datasets 2 and 3)and density modification using RESOLVE (29). To obtain thenative PI31 FP domain structure free of introduced mutations,a partial model was built into this map and was subsequentlypositioned by molecular replacement into the native unit cell(dataset 1). Refinement against this native dataset collected inhouse produced the final model (Table 1, dataset 1). The finalmodel lacks 9 residues from the C termini of both copies (chainA and B) and residue Ser-75 in chain A. Residues in loop 72–76and 96–98 are relatively mobile as reflected in high tempera-ture factors and poor electron density. ChainA andB are essen-tially identical (root mean square deviation of 0.419 Å over 141C� residues). Four of the cysteine residues in each chain appearto have undergone partial oxidation and have been modeled assulfenic acids. Coordinates have been deposited in the ProteinData Bank data base with accession code 2VT8.Isothermal Calorimetry—Isothermal calorimetry experi-

ments used a MicroCal VP-ITC according to manufacturer’sinstructions. All proteins analyzed by isothermal calorimetry(ITC) were purified by size exclusion chromatography and dia-lyzed into the same buffer (20 mM Hepes, pH 8, 50 mM NaCl,and 1 mM �-mercaptoethanol) before measurement of theirmolar concentration. The Fbxo7�Skp1 concentration withinthe sample cell was 20 �M, and the PI31 titrant concentrationwas 100 �M. The experiments were carried out at 30 °C with 28injections each of 10 �l measured with stirring, and all settingswere constant for both wild type and mutant experiments.Results were analyzed using theORIGIN7 software provided byMicroCal and using a one-binding-site model.Analytical Ultracentrifugation—For analytical ultracentrifu-

gation (AUC) experiments, recombinant proteins were storedand analyzed in a buffer containing 20 mM Tris-HCl, pH 8, 50mMNaCl, and 5mM �-mercaptoethanol. A BeckmanXL-I ana-lytical ultracentrifuge was used to measure both absorbanceand interference data according to the manufacturer’s instruc-

Structural and Functional Link between Fbxo7 and PI31

22326 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 32 • AUGUST 8, 2008


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

tions. Protein parameters used for data analysis and the calcu-lation of initial experimental parameters were calculated usingthe program SEDNTERP (30). Data were analyzed using theAUC machine software and the programs SEDFIT andSEDPHAT (31) using the model monomer-dimer equilib-rium in the final analysis.Yeast Two-hybrid Screens—Fbxo7 129–522 and 129–398

were subcloned into pGBDU-C1 and co-transformed withlibrary plasmid (Clontech) into PJ69–4a yeast two-hybridstrain (32).Cell Culture—Human osteosarcoma (U2OS) and Jurkat E6

cell lines were obtained from Cancer Research UK (LRI CellProduction). U2OS cells were grown in Dulbecco’s modifiedEagle’s medium, whereas Jurkat E6 cells were grown in RPMImedia. Both media were supplemented with 10% fetal calfserum (Helena Biosciences), 2 mM glutamine, 100 units/mlpenicillin, and streptomycin at 37 °C in a humidified 5% CO2atmosphere. U2OS cells were seeded the day before transfec-tion using FuGENE (catalog #1814443, Roche Applied Sci-ence). To visualize the subcellular localization of Fbxo7, cellswere transfected with a construct encoding a fusion of dsREDwith full-length Fbxo7. 24 h post-transfection, live cells werevisualized by confocal microscopy. For immunofluorescenceassays on PI31, cells were grown on glass coverslips, fixed in 3%paraformaldehyde, permeabilized with 0.1% Triton X, andblocked with 5% fetal bovine serum before incubation with pri-mary antibody against PI31 (1:200) and secondary rhodamine-conjugated anti-mouse antibody (1:500). The fluorescent dye3,3�-dihexyloxacarbocyanine iodide (DiOC6(3)) (Invitrogen)was included in the final wash to permit visualization of theendoplasmic reticulum by confocal microscopy. For co-immu-nofluorescence assays, cells were grown on glass coverslips,fixed in 70% ethanol, permeabilized with 0.1% Triton-X, andblocked with 0.1% gelatin before staining with affinity-purifiedFbxo7 polyclonal antibody (1:40) and PI31 monoclonal anti-body (1:10). Secondary antibodies sheep anti-rabbit conjugatedto AlexaFluor 488 (Molecular Probes) and goat anti-mouseconjugated to Cy5 (DAKO) were both used at 1:300. Coverslipsweremounted with Vectashield (Vector) mountingmedia con-taining 4�,6-diamidino-2-phenylindole. Cells were visualizedby epifluorescence microscopy.Fractionation, Immunoprecipitation, and Western Blotting—

U2OS cells were fractionated by resuspending in 100 mM Tris-HCl, pH 7.4, 100mMNaCl, 25mMMgCl2, and adding 40�g/mldigitonin and incubating on ice for 10min to lyse plasmamem-brane. Nuclei were separated from cytoplasmic fraction by cen-trifugation at 3000 rpm for 10min at 4 °C and lysed in radioim-mune precipitation assay buffer (50 mM Tris, pH 7.5, 150 mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS,protease inhibitor mixture). For immunoprecipitation, cellswere 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 phenyl-methylsulfonyl fluoride) and protease inhibitor mixture for 10min on ice before the addition of NaCl to a final concentrationof 150 mM. Lysates were centrifuged for 15 min at 13,000 rpmand then immunoprecipitated with anti- FLAG-M2-agaroseslurry for 3 h at 4 °C on a rotating wheel. Agarose beads werethen washed 4 times with NET2 buffer (50 mM Tris-HCl, pH

7.5, 150mMNaCl, and 0.05% Triton X-100) before being resus-pended in 4� Laemmli buffer and resolved by SDS-PAGE. Thefollowing antibodies were used for immunoprecipitation orWestern blotting: Cdk6 (SC177) (Santa Cruz BiotechnologyInc.), FLAG (F3165, Sigma), and T7 (catalog #69522-3, Nova-gen). The antibody against Fbxo7 has been previously described(13). The monoclonal antibody raised against the FP domain ofPI31 was made by Monoclonal Antibody Service, CancerResearch UK. Anti-rabbit IgG and anti-mouse IgG antibodiesconjugated to horseradish peroxidase were obtained fromSanta Cruz Biotechnology, Inc. and Jackson ImmunoResearchLaboratories.

RESULTS

Mapping Protein Interaction Sites within Fbxo7 and Identifi-cation of PI31 as an Fbxo7 Binding Partner—To map regionswithin Fbxo7 responsible for mediating its in vivo interac-tions with known protein partners, we prepared several dele-tion constructs based on predicted domain boundaries ofFbxo7 and incorporated an N-terminal T7 epitope. Theseincluded constructs lacking the N-terminal Ubl domain, theF-boxmotif, or the C-terminal PRR (Fig. 1A).We first soughtto map the binding site for Cdk6, a validated binding partnerfor Fbxo7 (13). Cdk6 was efficiently co-immunoprecipitatedby full-length Fbxo7-(1–522) and by a mutant of Fbxo7 lack-ing the F-box domain (�F-box) indicating that the interac-tion did not require the F-box (Fig. 1, A and B). We nextdeleted the first 129 amino acids of Fbxo7 which includes theUbl domain, designated as Fbxo7-(129–522). However, bySDS-PAGE, this mutant co-migrated with IgG heavy chain(molecular mass, 50 kDa), making it unusable in our co-immunoprecipitation assays. Fbxo7-(129–398) (lackingboth the Ubl domain and the PRR region) was also able toco-immunoprecipitate Cdk6 in vivo (Fig. 1, A and B), show-ing neither region was essential for binding. Because Fbxo7-(129–398) was predicted to have an unstructured sequenceof 40 amino acids at the N terminus (129–169), we therefore,truncated the construct further to produce Fbxo7-(169–398). This protein was now unable to bind Cdk6 (Fig. 1B),indicating that within the context of Fbxo7-(129–398),amino acids 129–169 were necessary to bind Cdk6.We then made further deletions from the N terminus

including Fbxo7-(239–522), which surprisingly, was able tobind Cdk6 despite the absence of residues 129–169. Thisindicated another region within 239–522 could mediateCdk6 interaction. This region contains the F-box domainand the PRR sequence. We deleted the unstructured C ter-minus of Fbxo7, creating Fbxo7-(239–381) and alsoattempted to express the C terminus, Fbxo7-(419–522), butthis protein could not be produced (Fig. 1, A and B). Fbxo7-(239–381) did not interact with Cdk6, suggesting that thesequences from 381–522, which includes the PRR, previ-ously proposed to mediate substrate recognition for HURPand c-IAP, could also contribute to Cdk6 interaction (17,18). Our data indicate that two regions from Fbxo7 can inde-pendently bind to Cdk6, suggesting Fbxo7 has a bipartiteCdk6 binding site. By mapping the binding site of a knownFbxo7-interacting partner to domains within Fbxo7, we val-




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

idated the use of this panel of Fbxo7-truncated constructs tosearch for new protein partners of Fbxo7 and, thus, forSCFFbxo7.Closer examination of Fbxo7 sequence conservation and

predicted secondary structure identified a putative globulardomain (residues 180–324) that precedes the F-box and fol-lows the Ubl and the N-terminal Cdk6 binding sequence. Wealso identified a highly conserved R(Ar)DP motif (where Arindicates any aromatic amino acid) within residues 466–496 ofthe PRR, with an as yet undetermined function. To investigatewhether residues 180–324 could function as a protein interac-tion module and to identify putative partner proteins, we pre-pared GST-tagged recombinant Fbxo7-(129–398)�Skp1 com-plexes for ex vivo affinity pulldown experiments from Jurkat cell

lysates as described under “Experimental Procedures” (33).Skp1 was co-expressed to engage the F-box motif and was usedas a positive control to demonstrate proper folding of Fbxo7(Fig. 1C). After incubation of recombinant GST-Fbxo7-(129–398)�Skp1 immobilized on glutathione-Sepharose beads withthe Jurkat cell lysate, the beads were washed and treated withthrombin to release Fbxo7-(129–398)�Skp1. Co-eluting pro-teins were then identified by mass spectrometry. Using thisapproach we identified PI31 (proteasome inhibitor 31kDa) asco-eluting with Fbxo7�Skp1 (Fig. 1C) but not from a GST con-trol elution. In addition, the known Skp1-binding protein Cul-lin1 and endogenous Skp1 also co-eluted with Fbxo7�Skp1. Thelatter was present as excess recombinant Fbxo7 was present inthe protein preparations.

25

37

50

IP cdk6total lysate

129-398

169-398

129-398

169-398

IP cdk6total lysate

75

50

vector

1-522

∆F box

vector

1-522

∆F box

37

IP cdk6total lysate

vector

239-522

vector

239-522

IP cdk6

15

total lysate

vector

239-381

419-522

vector

239-381

419-522

A

B

SC-Ura-Leu SC-Ura-Leu-His-Ade

vector+ PI31

Fbxo7 129-522+ PI31

HVS cyclin+ PI31

Fbxo7 129-398+ PI31

Fbxo7 129-522+ PI31

Fbxo7 129-398+ PI31

HVS cyclin+ PI31

vector+ PI31

C D

Cullin1

PI31

Skp1

Skp1(∆H8)

Actin

PI31(truncated)

Fbxo7(129-398)

97

66

45

30

20

GST GST-Fbxo7(129-3

98)

-Skp1(∆H8)

Fbxo7

+

+

+-

∆F box

129-398 169-398 239-522

Ubl F box Proline rich

33278 129 1691 522375 423

cdk

R(Ar)DP466 496

1-522+

419-522nd- 239-381

129-522nd

FIGURE 1. Mapping Fbxo7 protein interaction sites. A, schematic of motifs within Fbxo7 and the deletion constructs used for co-immunoprecipitations withCdk6. �, detectable Cdk6 binding; �, no detectable Cdk6 binding; nd, not determined. B, in vivo co-immunoprecipitation (IP) assays using Fbxo7 deletionmutants. Lysates were immunoprecipitated with antibodies to Cdk6 and analyzed for the associated proteins as indicated. C, Coomassie-stained SDS-PAGE gelof binding proteins from Jurkat cell lysates isolated by affinity-tagged recombinant Fbxo7-(129 –398)�Skp1. Proteins were identified by mass spectrometry. D,yeast-2-hybrid assay for proteins interacting with the indicated bait proteins. Yeast cells were transformed as indicated (pGBD bait � pGAD prey) and platedon media selecting for the plasmids (SC-Ura-Leu) and on media selecting for activation of reporter genes (SC-Ura-Leu-His-Ade).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

In parallel to the affinity pulldown experiments, we under-took yeast two-hybrid screens of a human cDNA library usingFbxo7-(129–398) and Fbxo7-(129–522) as bait (Fig. 1D). Inboth screens more than 60% of the library plasmids that wereisolated had PI31 fused with Gal4 activation domain (GAD),whereas 7% of the clones contained Skp1. Assaying for activa-tion of theHIS3 and ADE2 reporter genes tested the specificityof the pGAD-PI31 interaction with the pGBD-Fbxo7 bait plas-mid. Yeast co-transformed with Fbxo7 plasmid and pGAD-PI31 grew on media lacking histidine and adenine. However,yeast transformed with either plasmid alone or a different baitplasmid encoding a viral cyclin fused to GBD together withpGAD-PI31 failed to grow under the same conditions (Fig. 1D).Taken together, the results from these independent screens andaffinity pulldown experiments indicate that PI31 can interactwith Fbxo7 and is a previously uncharacterized binding partnerfor Fbxo7.Fbxo7 and PI31 Are Structurally Related—After the identifi-

cation of PI31 as a putative Fbxo7 binding partner, we analyzedsequences of Fbxo7 and PI31 and noticed that these proteinsare related at both a sequence and structural level despite theirinvolvement in quite different multiprotein complexes. Bothcontain a domain equivalent to residues 180–324 of Fbxo7 andan unstructured C-terminal rich in proline (Fig. 2A). Thesequences of Fbxo7 (residues 180–324) and PI31 (residues1–151) are 24% identical and 45% similar (Fig. 2B). Furthersearches with sequence databases found no further examples ofthis domain, which we refer to as the FP domain (Fbxo7 andPI31). We also found that the R(Ar)DP motif originally identi-

fied in the PRRof Fbxo7 is absolutely conserved in the PRRof allPI31 sequences identified to date (Fig. 2C). Thus, Fbxo7 andPI31 share a structural and evolutionary relationship despitetheir distinct biological functions.The FP Domain Adopts an �/�-Fold—We then determined

the crystal structure of the human PI31 FP domain at 2.64 Å bySAD phasing (Table 1). Experimental phases were obtainedusing data collected from crystals of selenomethionine-labeledL7M mutation as described under “Experimental Procedures.”Representative electron density for the refined structure isshown in Fig. 3A. The FP domain has approximate dimensionsof 40 � 28 � 25 Å and consists of an �/�-fold with a centralfive-stranded anti-parallel�-sheet flanked by twoN-terminal�helices and three C-terminal � helices (Fig. 3B). All the helicespack against one face of the �-sheet, leaving the opposite sidemore accessible to solvent. Helix �2 is almost completely bur-ied within the FP domain and contains two highly conservedcharacteristic residues, Asp-20 and His-27 (Fig. 2, B and C).This DX7H motif is intimately associated with both tyrosineresidues on the YXLXYmotif of strand�2 (residues 62–71) andcontributes to a network of conserved hydrogen bonds. Theseinclude hydrogen bonds from His-27 side chain to both theTyr-69 hydroxyl and to the main-chain carbonyl of Glu-52;OD1 of Asp-20 hydrogen bonds to Tyr-65 hydroxyl and theNZatom of Lys-62, whereas OD2 hydrogen bonds to the main-chain amide functions of Thr-16 and Cys-17. A further featureof the FP domain structure is a striking hydrophobic patchcomprising Leu-64, Ile-83, Val-85, and Ile-90 on the exposedsurface of the central�-sheet, which is discussed later (Fig. 3D).

PI31

Fbxo7

232 242 252

Pan_troglodytes_PI31 DP P RF GP .....LPPGAV PGA F . .IGTSPPG..Pongo_pygmaeus_PI31 DP P RF GP .....LPPGAV PGA F . .IGTSPPG..Homo_sapiens_PI31 DP P RF GP .....LPPGAV PGA F . .IGTSPPG..Rattus_norvegicus_PI31 DP P RF GP .....LPPGAV PGA F . .IGTSPSG..Mus_musculus_PI31 DP P RF GP .....LPPGAV PGA F . .IGTSPSG..Xenopus_laevis_PI31 DP P RF GP .....LPPGAV PGA F . .IGSGRPR..Gallus_gallus_PI31 DP P RF GP .....LPPGAV PGA F . .LGAGRAG..Apis_mellifera_PI31 DP P RF GP .....LPSGAV PFA F P DLDRPRPR..Drosophila_melanogaster_PI31 DP . RF NP .....M..GPG PVP F . .LNPNRPGQ.Drosophila_pseudoobscura_PI31 DP I RF GP .....MP.GNG SYP F . .IDPNLPNR.Arabidopsis_thaliana_PI31 DP P RF GP .....PHPGMP PGA Y P GVPGFEPGRFOryza_sativa_PI31 DP P RY GP .......PGSV PGG I P DVPGFEPSRFPlasmodium_yoelii_Fbxo7 DP P RY GP ........... QKL I . ..FGNEP...Plasmodium_berghei_Fbxo7 DP P RY GP ........... QNL I . ..FGNEP...Pongo_pygmaeus_Fbxo7 DP P RF GP PGPGETPSQFP LRP I . .LPGPNPILPMacaca_fascicularis_Fbxo7 DP P RF GP PGPGETPSQFP LRP I . .LPGPNPILPHomo_sapiens_Fbxo7 DP P RF GP PGPGETPSQFP LRP V . .LPGPNPILPBos_taurus_Fbxo7 DP P RF GP PGPGETPSQFP LRP V . .LPGPNPILPCanis_familiaris_Fbxo7 DP P RF GP PGPGETPSQFP LRP V . .LPGPRPTLPGallus_gallus_Fbxo7 DP P HF GS PGPGEAPGQFP FRP I . .LPGANPTLPMus_musculus_Fbxo7 DP P RF DP PRPGELPGQFR LRP V . .LPGPHSLLP

C

B 1 10 20 30 40 50 60 70

Homo_sapiens_PI31 T LV LH V Y W YVL Y ...........MAGLEVLFASAAPAI CRQDA CF WEV THG FGLGVGDQPGPNDKK SELLPAG NNNKDL R EHomo_sapiens_Fbxo7 MLCSESVEGQVPHSLETLYQSAD..C DANDA VL LLM ESG IPQG.TEAKALS... ...MPEK KLSG.V Q M

80 90 100 110 120 130 140

Homo_sapiens_PI31 IL I T S L V II IKDGSRKL.LVKAITVESSM NV.LEYGS..QQVADLTLNLDDY DAEHL. .GDFHR YKN EE RSRI SG TP Homo_sapiens_Fbxo7 VV I I L L K LV LPLCEGSSATLTCVPLGNLI NATLKINNEIRSVKRLQLLPESF CK.EKL GENVAN YKD QK SRLF DQ YP

α1 α2 2ß1ß

ß3 ß4 ß5 α3 α4

W YKL YS LI IH L YYH

151 1720811PI31

232 254

Fbxo7

A

hcir enilorPxob FlbU

522423

cdk

R(Ar)DP466 496423081

FP domain

Proline rich

R(Ar)DP

FP domain

FIGURE 2. Sequence and structural conservation between Fbxo7 and PI31. A, schematic of the similarity between Fbxo7 and PI31 proteins, including theFP domain, the PRR, and the conserved R(Ar)D motif. B, FP domain sequences for human Fbxo7 and PI31. Sequence numbers are taken from the Homo sapiensPI31. Conserved residues across both PI31 and Fbxo7 families are indicated in yellow, and identities are in red (38). Residues targeted by mutation in this studyare indicated by a red star above the sequence. Secondary structure from the PI31 FP domain structure is indicated above the sequence. C, sequence conservationwithin the C-terminal R(Ar)D motif of PI31 and Fbxo7.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

An irregular �1-�2 loop unique to PI31 meanders alongsideand shields the �1 edge strand from solvent and is stabilized bya salt bridge formed by residues Asp-48 and Arg-68.Searches with secondary structure matching failed to reveal

any other protein with a topology exactly matching that of theFP domain. A remote similarity was found with the E. coli pro-tein CyaY (ProteinData Bank code 1EW4) (34), which is closelyrelated to frataxin (Fig. 3E). CyaY and the PI31 FP domainsuperpose with a root mean square deviations of 2.85 Å (68 C�atoms). Although topologically similar with a single anti-paral-lel �-sheet and a helix equivalent to �2, their respective hydro-phobic cores are quite distinct, indicating that no evolutionaryrelationship is likely.Dimeric Arrangement of the PI31 FP Domain in Solution and

within Crystals—Characterization of recombinant PI31 FPdomain in solution by size exclusion chromatography indicatedthat the domain migrated with an apparent molecular mass of32 kDa, consistent with a dimeric structure (Fig. 4A). Sedimen-tation equilibrium analysis by analytical ultracentrifugationunder physiological buffer conditions also indicated a mono-mer-dimer equilibrium with an affinity constant (Kd) of 4.85�M. Two distinct dimeric arrangements of the PI31 FP domainare presentwithin the crystal lattice, bothwith sufficiently largeburied surfaces to be candidates for the solution dimer inter-face. One surface involves predominantly hydrophobic resi-dues from helix �1 centered on Val-6 and buries an area of557.2 Å2 per protomer (Fig. 4, A, right panel, and B). Theseresidues are generally conservedwithin PI31 sequences, andwerefer to this as the “helical interface” (Fig. 4B). A second dimericcontact involves the solvent-accessible surface of the �-sheet,which is also relatively hydrophobic and covers 279.9 Å2 perprotomer (Fig. 4A, left panel). This “� interface” is centered onresidues Ile-83 and Ile-90 (Fig. 3D and 4B). Each of the twomolecules within the asymmetric unit forms equivalent crystal-

lographic dimers, suggesting these arrangements are not solelya result of lattice contacts.The PI31 FP Domain Homodimerizes through a Helical

Interface—To determine which of the two interfaces wasrequired for PI31 homodimerization, disrupting substitutionswere introduced into the dimeric interface of both the helical(V6R) and the� interface (I83E/I90E) (Fig. 4,A andB). Both theV6R and I83E/I90E mutants expressed at similar levels to thewild type protein and purified under identical conditions (datanot shown). We probed the oligomeric state of the mutatedPI31 proteins by size exclusion chromatography andAUC.V6Rmigrated with amolecular weight by size exclusion chromatog-raphy consistent with a monomer (Fig. 4B), and AUC datacould not be fitted with a monomer-dimer equilibrium model,indicating that this mutant is monomeric. In contrast, the dou-ble mutant I83E/I90E eluted at the same volume as wild typePI31 by size exclusion chromatography, and AUC gave a calcu-lated Kd of 3.95 �M similar to the wild type PI31, indicating thedimeric structure was not perturbed by these point mutations.The monomeric nature of the V6R mutant demonstratesthat the PI31 FP domain homodimerizes via the helical inter-face, the larger of the two buried surfaces observed withinthe crystal lattice.The FP Domain of Fbxo7 and PI31 Heterodimerize through a

� Interface—We investigated whether the � interface of thePI31 FP domain was involved in mediating heterodimerizationwith the Fbxo7 FP domain by using GST-Fbxo7-(169–398)�Skp1 complex containing the Fbxo7 FP domain. The affin-ity of the FP domain of PI31 for Fbxo7-(169–398)�Skp1�H8was measured by ITC (Fig. 4C). The assumption that there isone binding site on Fbxo7�Skp1 per PI31 protomer gave the bestfit to the experimental data. A Kd of 0.37 �M (error 12%) wasdetermined for the binding of PI31 FP domain to Fbxo7-(169–398) �Skp1�H8. This is an �10-fold higher affinity than that

TABLE 1Values in parenthesis correspond to the highest resolution shell

Sample Native PI31(residues 1–151)

L7M PI31(residues 1–151)

L7M PI31(residues 1–151)

Dataset 1 2 3Diffraction data Native Selenomethionine SelenomethionineSpace group C2 C2 C2Cell constants (Å; o) a � 109.5, b � 45.2 a � 108.4 , b � 42.8, a � 108.7, b � 42.3,

c � 67.3; � � 111.5o c � 66.6; � � 109.1o c � 66.3; � � 109.4oZa 2 2 2Wavelength (Å) 1.541 0.97855 (peak) 0.97855 (peak)Resolution (Å) 28.1-2.64 (2.74-2.64) 39.75-2.5 (2.64-2.5) 27.12-2.50 (2.64-2.5)Completeness (%) 99.6 98.4 99.9Multiplicity 7.1 (6.5) 7.3 (7.4) 7.2 (7.3)Rmeas (%) 7.6 (38.8) 7.2 (16.9) 4.7 (15.1)Rp.i.m.a (%) 3.2 (15.4) 3.3 (9.5) 2.4 (6.1)Rano(%) 4.5 (8.0) 4.7 (8.4)�I/sd 21.1 (4.8) 25.5 (12.5) 31.9 (11.8)

RefinementRfact(%) 20.2 (29.5)Rfree(%) 25.3 (40.0)Reflections 9115 (648)Number of protein atoms 3144Number of solvent atoms 36Wilson B factor 38Average B factor (Å2) 42.8Root mean square deviation bonds (Å),

angles (°)0.024, 2.262

Ramachandran plot (%) (core, allowed,generously allowed, disallowed)

85.1/12.4/0.8/1.6

a Rp.i.m. is the precision R-factor.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

determined for the PI31 homodimer. Similar ITC experimentsusing the dimeric PI31 I83E/I90E double mutant showed nodetectable binding as isotherms were identical to diluting sam-ple with buffer (Fig. 4C). These in vitro data indicated that the�interface of the PI31 FP domain forms a dominant part of theheteromeric interaction with Fbxo7�Skp1.To test this interaction in vivo, double point mutations I83E/

I90E were engineered into full-length of PI31 tagged at the Nterminus with a FLAG epitope and expressed in U2OS cells.

Immunoprecipitates from these cell lysates using anti-FLAGantibodies were subsequently analyzed byWestern blotting forthe presence of Fbxo7. Endogenous Fbxo7 co-immunoprecipi-tatedwithwild type PI31; however, despite expression at equiv-alent levels, the I83E/I90E mutations in PI31 did not interactwith Fbxo7 in vivo (Fig. 4D). We noted that these point muta-tions reduced themobility of PI31 comparedwith thewild type.We also determined whether equivalent mutations within

the FP domain of Fbxo7 would ablate its heterodimerization

A

α1

α4α3

α2α5

α1

α4α3

α2α5

β1

β2

β3 β4 β5

β1

β2

β3 β4 β5B

α2

β2

β3

Tyr69 His27

Leu67

Asp20

Tyr65

DC

N

C

α1α2

α3

α5

ß1 ß2ß3

ß4

ß5

α4N

C

α1

α2

ß1ß2

ß3 ß4

ß5

ß6

C

E

Leu64

Ile83

Val85

Ile90

FIGURE 3. Structure of the PI31 FP domain. A, the final 2m Fo � D Fo electron density map from REFMAC, contoured at 1 � with the refined PI31 FP domainstructure. B, stereo representation of the PI31 FP domain with � strands in yellow and � helices in blue. C, interaction of the YXLXY motif on the �2 strand throughhydrogen bonds with the conserved residues Asp-20 and His-27 on the �2 helix. D, surface of the FP domain highlighting the hydrophobic patch on theexposed surface of the �-sheet. E, the PI31 FP domain (left panel) and CyaY (right panel), each colored from blue at the N terminus to red at the C terminus.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Dimer interacting through beta interface

Dimer interacting through helical interface

A

C

Void 66 43 30 12.4

Val6

Val6

PI31 (1-151) V6R

PI31 (1-151) wild type

PI31 (1-151) I83E, I90E

Ile90Ile83

Ile83Ile90

B

Titrant PI31 (1-151) PI31 (1-151)I83E/I90E PI31 (1-151)Cell Fbxo7 (169-398)-Skp1 Fbxo7 (169-398)-Skp1 Fbxo7 (169-398)(V253E)-Skp1

E

D

2520

50 vector

vector

WT

WT

V253E

V253E

V175R

V175RGSTGST-P

I31 GST GST-PI3110% input

vector

WT

V253E

V175R

75

IP FlagWB Fbxo7

total lysateWB Fbxo7

total lysateWB PI31

vector

PI31PI31 I8

3E/I90E

75

75

50

37

F

vector

vector

vector

WT

WT

WT

V175R

V175R

V175R

V253E

V253E

V253EGST

GST-Fbxo

7(129-3

98)

GST GST-Fbxo7(129-398)20% input

75

2520

50




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

with PI31. Based on structural align-ments of the two � interfaces, Val-253 in the FP domain of Fbxo7 isequivalent to Ile-83 in PI31 (Fig. 2B).V253E Fbxo7mutant was expressedand purified as a GST fusion proteinas outlined above. Fbxo7-(169–398V253E)-Skp1�H8 behaved simi-larly to the PI31 I83E/I90E mutantin ITC experiments (Fig. 4C), givingno detectable binding. Similarly, weused in vitro binding assays usingGST-PI31 to pull down in vitro tran-scribed and translated full-lengthT7-tagged Fbxo7 alleles (Fig.4E). This confirmed that Fbxo7-(169–398 V253E)-Skp1�H8 wasseverely impaired in binding PI31(Fig. 4E). We also tested whetherFbxo7�Skp1 is able to homodimerizeand whether V253E mutationaffects this interaction. IndeedFbxo7-(169–398)-Skp1�H8 doespull down wild type Fbxo7 but notFbxo7 (V253E) mutant (Fig. 4F).This indicates that Fbxo7 doesindeed homodimerize and that thisinteraction also involves the Fbxo7� interface centered on Val-253.Therefore, the binding site for PI31overlaps with a region of Fbxo7required for homodimerization.Wenote that as the in vitro Fbxo7expression and purification reliedupon co-expression of Skp1, a rolefor Skp1 in these assays cannot beformally excluded.PI31 and Fbxo7 Cellular

Localization—Ourdata suggest thatthe FP domains of PI31 and Fbxo7would be capable of interacting invivo, whichmay have functional sig-nificance, so we tested whether theyshare a subcellular location. WhenU2OS cell lysates were separatedinto nuclear and cytoplasmic frac-tions and probed for Fbxo7 andPI31, both proteinswere found to becytoplasmic, and a portion of Fbxo7

FIGURE 4. Characterization of interaction surfaces for the PI31 and Fbxo7 FP domains. A, superposition of gel filtration traces from size exclusionchromatography of PI31 FP domain wild type (pink), I83E/I90E (blue), and V6R (orange). Black arrows show the positions of molecular weight markers (inkDa). The y axis is absorbance at 280 nm, and the x axis is column volume. B, ribbon diagrams indicating the two distinct PI31 dimers within the crystallattice. C, ITC of PI31 and Fbxo7 wild type and mutant proteins. Left panel, wild type PI31 FP domain titrated into Fbxo7�Skp1; center panel, PI31(I83E/I90E) FP domain as titrated into Fbxo7�Skp1; right panel, wild type PI31 FP domain as titrated into Fbxo7-(V253E)-Skp1. D, co-immunoprecipitation(IP) of FLAG-tagged PI31 proteins with Fbxo7. Cells transfected with Fbxo7 and either wild type or mutant (I83E/I90E) PI31. Lysates were immunopre-cipitated with FLAG antibodies, and the presence of Fbxo7 was assayed by Western blotting (WB). E and F, in vitro binding assays using either GST,GST-PI31, or GST-Fbxo7-(129 –398) fusion proteins as indicated to pull down in vitro transcribed and translated full-length T7-tagged Fbxo7 alleles (wildtype (WT), V253E, or V175R). V175R was included as a control valine mutation outside of the FP domain. The left panel shows Coomassie-stainedSDS-PAGE of the GST proteins, and the right panel shows Western analysis for the T7 epitope on full-length Fbxo7 proteins.

PI31

Fbxo7

nuclear

cytoso

lic

A dsRED-Fbxo7B

D13IPegrem

Fbxo7 DAPI

E

PI31

Fbxo7

contro

l dsR

NA

Fbxo7 d

sRNA

ER (DiOC (3))6

PI31 mergeC

FIGURE 5. Localization of Fbxo7 and PI31. A, fractionation of Fbxo7 and PI31. Cells were separated intonuclear and cytosolic fractions and Western-blotted for the presence of proteins as indicated. B, localiza-tion of dsRED-Fbxo7 in the cytoplasmic compartment of live cells. U2OS cells were transfected with aplasmid expressing a fusion of Fbxo7 to dsRED and visualized by fluorescence microscopy. C, localizationof PI31. U2OS cells were fixed, and indirect immunofluorescence assays used a primary monoclonalantibody to PI31 and a secondary antibody conjugated to rhodamine (red) as probes. Cells were alsostained with a lipophilic dye DiOC6(3) (green). ER, endoplasmic reticulum. D, co-localization of a fraction ofendogenous Fbxo7 and PI31. An immunofluorescence assay for Fbxo7 (green) and PI31 (red) is shown;merged signals are yellow. Nuclei were stained with 4�,6-diamidino-2-phenylindole (DAPI, blue). E, reduc-tion of Fbxo7 protein does not affect the levels of PI31. Western blotting for endogenous Fbxo7 and PI31in lysates of cells transfected with control double-stranded (ds) RNA or dsRNA against Fbxo7. The solidarrow indicates Fbxo7; the dashed arrow is a cross-reacting band.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

was present in the nucleus (Fig. 5A). In live U2OS cells, Fbxo7also localized predominantly within the cytosol, with a smallproportion localized in the nucleus, in agreement with the frac-tionation (Fig. 5B). The subcellular localization of PI31 wasinvestigated using an antibody generated against the FP domainof PI31. Immunofluorescence assays on fixed U2OS cells dem-onstrated that the majority of PI31 was perinuclear and co-localizedwith a lipophilic dye that stains the endoplasmic retic-ulum (Fig. 5C).We also found that detectable amounts of Fbxo7and PI31 overlap inmerged images indicating they can be local-ized to discrete subcellular compartments (Fig. 5D).Because Fbxo7 is a subunit of an SCF-type E3 ubiquitin ligase

and it co-localizes in immunofluorescence assays and co-im-munoprecipitates with PI31, we testedwhether reducing Fbxo7affected PI31 levels. U2OS cells were transfected with controldouble-stranded (ds)RNA or dsRNA against Fbxo7, as previ-ously described (13), and cell lysates were analyzed byWesternblotting for the presence of endogenous Fbxo7 and PI31. PI31levels, however, were unaffected by decreases in Fbxo7 levels,suggesting that it does not precipitate the ubiquitin-mediateddegradation of PI31 (Fig. 5E).

DISCUSSION

To understand how F-box proteins engage SCF substratesand other potential regulatory proteins through protein-pro-tein interaction, we focused our analysis on the Fbxo7 subunitof the SCFFbxo7. This SCF-type E3 ubiquitin ligase is reported topromote the ubiquitin-mediated degradation of HURP andc-IAP (17, 18), and the Fbxo7 subunit acts as a specific enhancerof cyclin D-Cdk6 complexes (13, 35). To identify further puta-tive substrates and protein-interactions, we combined struc-tural, biochemical, and genetic approaches to study regions ofFbxo7 required for protein interaction. Previously publisheddata implicated the C-terminal PRR as being required for bind-ing to HURP and c-IAP (17, 18). We find that two separateepitopes on Fbxo7 are required for its in vivo interaction withCdk6, one within amino acids 129–169 and the second span-ning the C-terminal PRR. This suggests that Fbxo7 has a bipar-tite interaction with Cdk6.We have identified a globular domain of �150 amino acids,

defined here as the FP domain, located between the Ubl andF-box of Fbxo7. Using affinity purification coupled with massspectrometry and yeast two hybrid techniques, we separatelyidentified PI31, a regulator of proteasome assembly, as interact-ing with Fbxo7�Skp1and also having an FP domain. These twoproteins have not been previously linked. Furthermore, PI31and Fbxo7 share not only an FP domain in common but also ahighly conserved R(Ar)DP motif embedded near the proline-rich C terminus. Fbxo7 appears to be an elaborated variant ofPI31, possessing additional domains (Ubl and F-box), whichpresumably provide additional functionalities through regula-tory and protein interaction sites.The PI31 FP domain structure reveals an �/�-fold with no

close structural relatives to date. The domain associates as adimer both in solution and within crystals. By structure-guidedmutation of surface residues, we found that the PI31 FP domainhomodimerizes through a predominantly helical interface andheterodimerizes with the Fbxo7 FP domain through contacts

between their � sheets. Previous studies fromMcCutchen-Ma-loney (21) showed that full-length PI31 forms homodimerswith an apparent Kd of 6.25 �M. Our determination of a Kd of4.85 �M for the isolated FP domain suggests that PI31 dimer-ization ismediated primarily through its FP domain. Size exclu-sion chromatography of cell lysates indicated that most of thePI31 in cells was present in complexes of �60 kDa, which isconsistent with dimer formation (data not shown). Whetherthe dimeric organization of PI31 is important for its inhibitoryfunction of immunoproteasome assembly is unclear at present.We also demonstrate that Fbxo7 andPI31heterodimerize via

a �-sheet surface on their FP domains and with an apparentaffinity constant of 0.37 �M. In vivo, only a fraction of the twoproteins occupy the same subcellular compartment. This didnot exclude the possibility that a proportion of either proteincould associate transiently and may lead to ubiquitin-protea-some system-mediated degradation. We, therefore, testedwhether reducing Fbxo7 protein expression levels by RNA-me-diated interference affected the levels of PI31 protein; however,no changes were observed, which argues against PI31 being adirect substrate for an SCFFbxo7 E3 ligase. Alternatively, thisinteraction may be relevant in different cell types or under dif-ferent growth conditions (e.g. cell stress or viral infection).

Both Fbxo7 and PI31 associate as homodimers. This oligo-meric state may be functionally relevant. For example, otherF-box proteins, such as Fbw7/Cdc4, �-TrCP, and Pop1/Pop2homologues, have D box domains thatmediate homodimeriza-tion, and the structure of one of these domains has beenrecently solved (36). D boxes are small 45-amino acid motifsclosely juxtaposed to the F-box domain, analogous to the FPdomain of Fbxo7. Dimerization of SCFCdc4 has been shown tobe required for its in vivo function andmay affect efficient ubiq-uitin chain elongation rather than substrate binding per se (10,36). Similarly FP domain-mediated dimerization of SCFFbxo7might also be required for E3 ligase function possibly by ena-bling cyclinD-Cdk6 complex formation. The overlap betweenthe PI31 binding site on Fbxo7 and Fbxo7 homodimerizationsurface suggests an intriguing possibility that PI31 could mod-ulate SCFFbxo7 function by antagonizing Fbxo7 homodimeriza-tion, a model we are actively exploring.This study and others have presented evidence that the C

terminus of Fbxo7 mediates binding to its substrates (17, 18),and the ability of PI31 to bind and inhibit proteasome activityhas also beenmapped to theC terminus (21).Within this regionis the most highly conserved sequence between the two pro-teins, the R(Ar)DP motif. Although its function is undeter-mined, the R(Ar)DP motif in PI31 partly overlaps a PPGXRconsensus binding site. In fact, PI31 has two such consensussites in its C terminus. These act as binding sites for GYFdomains, which facilitate protein interactions with proline-richsequences (37). Fbxo7, however, does not contain the PPGXRmotif, suggesting theR(Ar)DPderives froma commonancestorof Fbxo7 and PI31 before their divergence to evolve differentbiological roles. Furthermore, Ubl domains, such as that foundat the N terminus of Fbxo7, have also been reported to bindto ubiquitin receptors, including subunits of the proteasome(16), providing a possible functional link with PI31 as both




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

proteins are capable of directly interacting with the protea-some (19–21).In conclusion, we havemapped regions required for protein-

protein interaction within Fbxo7 and identified a novel dimer-ization domain that is also present within the structural-relatedPI31. The proposed link between Fbxo7 and PI31 at both astructural and functional level is further consolidated by theobserved direct interaction between these proteins. Site-spe-cific substitutions within both proteins ablate this interaction,and this knowledge can be exploited in vivo to probe the func-tion of these proteins, their interaction, and the functional sig-nificance of their respective dimeric arrangements.

Acknowledgments—Mass spectrometry was carried out by the Pro-tein Analysis Laboratory at the London Research Institute. Wegratefully acknowledge help from Jane Sandall and SarahWestcottin using AUC and ITC.

REFERENCES1. Hershko, A. (1997) Curr. Opin. Cell Biol. 9, 788–7992. Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67,

425–4793. Hicke, L., and Dunn, R. (2003) Annu. Rev. Cell Dev. Biol. 19, 141–1724. d’Azzo, A., Bongiovanni, A., and Nastasi, T. (2005) Traffic 6, 429–4415. Nakayama, K. I., and Nakayama, K. (2005) Semin. Cell Dev. Biol. 16,

323–3336. Petroski,M. D., andDeshaies, R. J. (2005)Nat. Rev.Mol. Cell. Biol. 6, 9–207. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., and Elledge,

S. J. (1996) Cell 86, 263–2748. Cenciarelli, C., Chiaur, D. S., Guardavaccaro, D., Parks,W., Vidal, M., and

Pagano, M. (1999) Curr. Biol. 9, 1177–11799. Winston, J. T., Koepp, D. M., Zhu, C., Elledge, S. J., and Harper, J. W.

(1999) Curr. Biol. 9, 1180–118210. Orlicky, S., Tang, X., Willems, A., Tyers, M., and Sicheri, F. (2003) Cell

112, 243–25611. Hao, B., Oehlmann, S., Sowa, M. E., Harper, J. W., and Pavletich, N. P.

(2007)Mol. Cell 26, 131–14312. Hao, B., Zheng, N., Schulman, B. A., Wu, G., Miller, J. J., Pagano, M., and

Pavletich, N. P. (2005)Mol. Cell 20, 9–1913. Laman, H., Funes, J. M., Ye, H., Henderson, S., Galinanes-Garcia, L., Hara,

E., Knowles, P., McDonald, N., and Boshoff, C. (2005) EMBO J. 24,3104–3116

14. Ilyin, G. P., Rialland, M., Pigeon, C., and Guguen-Guillouzo, C. (2000)Genomics 67, 40–47

15. Kelley, L. A., MacCallum, R. M., and Sternberg, M. J. (2000) J. Mol. Biol.299, 499–520

16. Hicke, L., Schubert, H. L., and Hill, C. P. (2005)Nat. Rev. Mol. Cell. Biol. 6,610–621

17. Hsu, J. M., Lee, Y. C., Yu, C. T., andHuang, C. Y. (2004) J. Biol. Chem. 279,32592–325602

18. Chang, Y. F., Cheng, C. M., Chang, L. K., Jong, Y. J., and You, C. Y. (2006)Biochem. Biophys. Res. Commun. 342, 1022–1026

19. Chu-Ping, M., Slaughter, C. A., and DeMartino, G. N. (1992) Biochim.Biophys. Acta 1119, 303–311

20. Zaiss, D. M., Standera, S., Holzhutter, H., Kloetzel, P. M., and Sijts, A. J.(1999) FEBS Lett. 457, 333–338

21. McCutchen-Maloney, S. L., Matsuda, K., Shimbara, N., Binns, D. D.,Tanaka, K., Slaughter, C. A., and DeMartino, G. N. (2000) J. Biol. Chem.275, 18557–18565

22. Zaiss, D. M., Standera, S., Kloetzel, P. M., and Sijts, A. J. (2002) Proc. Natl.Acad. Sci. U. S. A. 99, 14344–14349

23. Schulman, B. A., Carrano, A. C., Jeffrey, P. D., Bowen, Z., Kinnucan, E. R.,Finnin, M. S., Elledge, S. J., Harper, J. W., Pagano, M., and Pavletich, N. P.(2000) Nature 408, 381–386

24. Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–26725. Collaborative Computational Project, Number 4 (1994) Acta Crystallogr.

D Biol. Crystallogr. 50, 760–76326. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997)Acta Crystallogr.

D. Biol Crystallogr. 53, 240–25527. Emsley, P., and Cowtan, K. (2004)Acta Crystallogr. D Biol. Crystallogr. 60,

2126–213228. Cowtan, K., and Main, P. (1998) Acta Crystallogr. D Biol. Crystallogr. 54,

487–49329. Terwilliger, T. C. (2000)Acta Crystallogr. D Biol. Crystallogr. 56, 965–97230. Cole, J. L., Lary, J.W.,Moody, P., and Laue, T.M. (2008)Methods Cell Biol.

84, 143–17931. Schuck, P. (2000) Biophys. J. 78, 1606–161932. James, P., Halladay, J., and Craig, E. A. (1996) Genetics 144, 1425–143633. Schneider, U., Schwenk, H. U., and Bornkamm,G. (1977) Int. J. Cancer 19,

621–62634. Cho, S. J., Lee, M. G., Yang, J. K., Lee, J. Y., Song, H. K., and Suh, S. W.

(2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8932–893735. Laman, H. (2006) Cell Cycle 5, 279–28236. Tang, X., Orlicky, S., Lin, Z., Willems, A., Neculai, D., Ceccarelli, D., Mer-

curio, F., Shilton, B. H., Sicheri, F., and Tyers, M. (2007) Cell 129,1165–1176

37. Kofler, M., Motzny, K., Beyermann, M., and Freund, C. (2005) J. Biol.Chem. 280, 33397–33402

38. Gouet, P., Courcelle, E., Stuart, D. I., and Metoz, F. (1999) Bioinformatics15, 305–308




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Lomonosov, El Kahina Meziane and Neil Q. McDonaldRebecca Kirk, Heike Laman, Phillip P. Knowles, Judith Murray-Rust, Mikhail

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

doi: 10.1074/jbc.M709900200 originally published online May 20, 20082008, 283:22325-22335.J. Biol. Chem.

10.1074/jbc.M709900200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/283/32/22325.full.html#ref-list-1

This article cites 38 references, 6 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M709900200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;283/32/22325&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/283/32/22325

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=283/32/22325&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/283/32/22325

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/283/32/22325.full.html#ref-list-1

http://www.jbc.org/

Documents

Structure of a Conserved Dimerization Domain within the F-box