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

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 SCFFbxo7 E3 ligase, and the proteasome inhibitor PI31. SCFFbxo7 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 α/β-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 SCFFbxo7. We present a model for FP domain-mediated dimerization of SCFFbxo7 and PI31. 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 SCFFbxo7 E3 ligase, and the proteasome inhibitor PI31. SCFFbxo7 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 α/β-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 SCFFbxo7. We present a model for FP domain-mediated dimerization of SCFFbxo7 and PI31. The levels of many regulatory and misfolded proteins are controlled by the ubiquitin-proteasome system (1Hershko A. Curr. Opin. Cell Biol. 1997; 9: 788-799Crossref PubMed Scopus (319) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). A series of enzymes termed E1 (ubiquitin-activating enzyme), E2 (ubiquitin carrier protein), and E3 6The abbreviations used are: E3ubiquitin ligaseHURPhepatoma up-regulated proteinIAPinhibitor of apoptosisITCisothermal calorimetrySCFSkp1-Cul1-F box proteinUblubiquitin-likePRRproline-rich regionFPFbxo7/PI31GSTglutathione S-transferase. (ubiquitin ligase) act in the ubiquitin-proteasome system as part of a concerted cascade to activate and conjugate ubiquitin via an isopeptide linkage to protein substrates (2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). Ubiquitin modification can affect the activity, localization, sorting, and stability of protein substrates (2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 3Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 4d'Azzo A. Bongiovanni A. Nastasi T. Traffic. 2005; 6: 429-441Crossref PubMed Scopus (202) Google Scholar). Recognition and recruitment of protein substrates to the ubiquitin-proteasome system machinery resides within the multisubunit E3 ubiquitin ligases, the largest group of which is the Skp1-Cullin1-F-box protein (SCF) family (1Hershko A. Curr. Opin. Cell Biol. 1997; 9: 788-799Crossref PubMed Scopus (319) Google Scholar, 5Nakayama K.I. Nakayama K. Semin. Cell Dev. Biol. 2005; 16: 323-333Crossref PubMed Scopus (292) Google Scholar, 6Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 9-20Crossref PubMed Scopus (1692) Google Scholar). All known SCF E3 ligases bind an E2 (ubiquitin carrier protein) enzyme through a RING finger-containing subunit (Rbx1) and utilize their F-box subunit to recruit substrates. Although classified by the presence of an F-box (Fbx) motif (7Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar), F-box proteins can be further divided into three classes depending on additional structural elements: Fbxw (WD40 motifs), Fbxl (with leucine-rich repeats), and Fbxo (F-box domain only) (8Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 9Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Recent structural data have shown how the Fbxw protein CDC4 (Fbw7) uses the center of the WD40 propeller to recognize the phosphorylated epitope of its substrate, cyclin E (10Orlicky S. Tang X. Willems A. Tyers M. Sicheri F. Cell. 2003; 112: 243-256Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 11Hao B. Oehlmann S. Sowa M.E. Harper J.W. Pavletich N.P. Mol. Cell. 2007; 26: 131-143Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Structural data also exist for the Fbxl protein Skp2 (Fbl1) as part of the SCFSkp2 complex bound to phosphorylated p27 (12Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20: 9-19Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). In contrast, little is known about how Fbxo proteins recognize their substrates or indeed whether other substrate-targeting domains exist within this subgroup. ubiquitin ligase hepatoma up-regulated protein inhibitor of apoptosis isothermal calorimetry Skp1-Cul1-F box protein ubiquitin-like proline-rich region Fbxo7/PI31 glutathione S-transferase. Fbxo7 has been characterized recently as a member of the Fbxo subgroup and is conserved among higher eukaryotes (8Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). As well as an F-box motif (residues 329-375), which interacts with Skp1 (8Cenciarelli C. Chiaur D.S. Guardavaccaro D. Parks W. Vidal M. Pagano M. Curr. Biol. 1999; 9: 1177-1179Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 9Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar, 14Ilyin G.P. Rialland M. Pigeon C. Guguen-Guillouzo C. Genomics. 2000; 67: 40-47Crossref PubMed Scopus (57) Google Scholar), Fbxo7 has an N-terminal ubiquitin-like (Ubl) domain (residues 1-78) (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar, 15Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1121) Google Scholar) (Fig. 1A), which is thought to mediate interactions with ubiquitin receptor proteins bearing ubiquitin binding domains (16Hicke L. Schubert H.L. Hill C.P. Nat. Rev. Mol. Cell. Biol. 2005; 6: 610-621Crossref PubMed Scopus (639) Google Scholar). The C terminus of Fbxo7 contains a proline-rich region (PRR) but lacks any predicted secondary structure. HURP (hepatoma up-regulated protein) and cIAP1 (an inhibitor of apoptosis protein) have both been reported as substrates for SCFFbxo7-mediated ubiquitination leading to proteasome-mediated degradation (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). Each binds to Fbxo7 through its C-terminal proline-rich region (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). Fbxo7 also acts as an assembly factor for cyclin D-Cdk6 complexes by virtue of its interaction with D-type cyclins and Cdk6 (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). In this study a combination of structural, biochemical, and genetic approaches were used to identify and characterize regions of Fbxo7 involved in protein interaction as a means to identify putative substrates of SCFFbxo7. Unexpectedly we uncovered a structural link between a domain from Fbxo7 and a related domain from PI31, a regulatory subunit of the immunoproteasome which is an in vitro inhibitor of the 20 S proteasome (19Chu-Ping M. Slaughter C.A. DeMartino G.N. Biochim. Biophys. Acta. 1992; 1119: 303-311Crossref PubMed Scopus (115) Google Scholar, 20Zaiss D.M. Standera S. Holzhutter H. Kloetzel P.M. Sijts A.J. FEBS Lett. 1999; 457: 333-338Crossref PubMed Scopus (77) Google Scholar, 21McCutchen-Maloney S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22Zaiss D.M. Standera S. Kloetzel P.M. Sijts A.J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). The crystal structure of this domain from PI31 a novel and We used this structure to the of to of the FP domain from PI31 for homodimerization and for interaction with We a for FP domain-mediated dimerization for SCFFbxo7 and PI31 and to Fbxo7·Skp1 complexes for the Skp1 and the C-terminal as B.A. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. 2000; PubMed Scopus Google Scholar). Fbxo7 (residues (residues and Fbxo7 (residues (residues were as The into Biochem. PubMed Scopus Google Scholar). The by from a a which a binding and into The into the Skp1 which lacks the C-terminal and B.A. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. 2000; PubMed Scopus Google Scholar). the system of PI31 were by and into the PI31 and and Fbxo7 and were the system All were by The were into and were in to an of protein by the of to a of for were by and in were from the or by and from the with PI31 and Fbxo7·Skp1 proteins were by and were and in 20 and by the into the E. on by protein and were a to the protein as and PI31 FP domain by in of protein in 20 and of well and within to FP domain and protein the as the were for of PI31 FP FP and the In a crystal to in a as a The of Biol. PubMed Scopus Google Scholar) were used for data and and were used for and model G.N. D. 1997; PubMed Scopus Google Scholar, P. K. Biol. 2004; PubMed Scopus Google in to the PI31 (residues PI31 (residues PI31 (residues is the of protein of factor is the in a to the structure data were to the of the from in PI31 FP domain not A by an leading to of and were the and The from by and used to with a of and of a of and of were by within K. P. Biol. 1998; PubMed Scopus Google Scholar) and with and and modification Biol. 2000; PubMed Scopus Google Scholar). the PI31 FP domain structure of a model into this map and by into the against this in the model The model lacks from the C of both A and and in A. in and are as in and A and are of of the in to have and have been as have been in the data with calorimetry used a to All proteins by isothermal calorimetry were by and into the and of their The Fbxo7·Skp1 within the 20 and the PI31 The were with of with and were for both and were the by and a proteins were and in a 20 and A used to both and data to the used for data and the of were the J.W. P. Cell Biol. PubMed Scopus Google Scholar). were the and the and P. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) the model in the and were into and with into P. J. 1996; PubMed Google Scholar). Cell and were from Cell were in were in were with and in a were the the of Fbxo7, were with a a of with were by on PI31, were on in with and with with against PI31 and secondary The L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google in the to of the by were on in with and with with Fbxo7 and PI31 to and to were both used were with were by and were by in and and on for to were from by for and in inhibitor were in and inhibitor for on the of to a of were for and with for were with and being in and by The were used for or Cdk6 and The against Fbxo7 has been (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). The against the FP domain of PI31 by and to were from and within Fbxo7 and of PI31 as an Fbxo7 map regions within Fbxo7 for mediating its in interactions with known protein we on predicted domain of Fbxo7 and an N-terminal the N-terminal the F-box or the C-terminal (Fig. We to map the binding for a binding partner for Fbxo7 (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar). Cdk6 by and by a of Fbxo7 the F-box domain that the interaction not the F-box (Fig. A and We the of Fbxo7 which the as by this with in both the domain and the also to Cdk6 in (Fig. A and region for predicted to have an of the terminus we the further to protein to bind Cdk6 (Fig. that within the of were to bind We further from the terminus which to bind Cdk6 the of region within mediate Cdk6 region contains the F-box domain and the We the C terminus of Fbxo7, and also to the C but this protein not be (Fig. A and not with that the from which the to mediate substrate for HURP and also to Cdk6 interaction (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). data that regions from Fbxo7 can bind to Fbxo7 has a Cdk6 binding the binding of a known partner to domains within Fbxo7, we the of this of to for protein of Fbxo7 for SCFFbxo7. of Fbxo7 and predicted secondary structure a putative domain (residues that the F-box and the and the N-terminal Cdk6 binding We also a conserved motif any within of the with an as whether as a protein interaction and to identify putative partner we complexes for from as U. G. J. 19: PubMed Scopus Google Scholar). Skp1 to the F-box motif and used as a to of Fbxo7 (Fig. of on with the the were and with to proteins were by this we PI31 inhibitor as with Fbxo7·Skp1 (Fig. but not from a In the known protein and Skp1 also with Fbxo7·Skp1. The present as Fbxo7 present in the protein In to the we of a and as (Fig. In both of the that were PI31 with domain of the for of the and the of the interaction with the with Fbxo7 and on and with or a a cyclin to with to the (Fig. the from and that PI31 can with Fbxo7 and is a binding partner for Fbxo7 and PI31 the of PI31 as a putative Fbxo7 binding we of Fbxo7 and PI31 and that proteins are related both a and structural their in complexes. a domain to of Fbxo7 and an C-terminal in (Fig. The of Fbxo7 (residues and PI31 (residues are and (Fig. with further of this which we to as the FP domain and We also that the motif in the of Fbxo7 is conserved in the of PI31 to (Fig. Fbxo7 and PI31 a structural and relationship their The FP an the crystal structure of the PI31 FP domain by were data from of as for the structure is shown in The FP domain has of and of an with a by N-terminal and three C-terminal (Fig. All the against of the the to is within the FP domain and contains conserved and (Fig. and motif is with both on the motif of (residues and to a of conserved from to both the and to the of of to and the of to the functions of and A further of the FP domain structure is a and on the of the which is (Fig. to PI31 and the from and is by a by and with secondary structure to any other protein with a that of the FP domain. A with the E. protein S.J. Lee Lee Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), which is related to (Fig. and the PI31 FP domain with a of Although with a and a to their are that relationship is of the PI31 FP in and within of PI31 FP domain in by that the domain with an of with a structure (Fig. by also a with an of of the PI31 FP domain are present within the crystal both with to be for the from on and an of (Fig. and are conserved within PI31 and we to this as the (Fig. A the of the which is also and (Fig. is on and (Fig. and Each of the within the are not a of The PI31 FP through a which of the required for PI31 were into the of both the and the (Fig. A and the and levels to the protein and not We the of the PI31 proteins by and with a by with a (Fig. and data not be with a that this is In contrast, the the as PI31 by and a of to the PI31, the structure not by The of the that the PI31 FP domain via the the of the within the crystal The FP of Fbxo7 and PI31 through a whether the of the PI31 FP domain involved in mediating heterodimerization with the Fbxo7 FP domain by complex the Fbxo7 FP domain. The of the FP domain of PI31 for by (Fig. The that is binding on Fbxo7·Skp1 PI31 the to the A of for the binding of PI31 FP domain to is an higher that for the PI31 the PI31 binding as were to with (Fig. in vitro data that the of the PI31 FP domain a part of the interaction with Fbxo7·Skp1. this interaction in mutations were into of PI31 the terminus with a epitope and in from were by for the presence of Fbxo7 with the mutations in PI31 not with Fbxo7 in (Fig. We that mutations the of PI31 with the We also whether mutations within the FP domain of Fbxo7 ablate its heterodimerization with PI31. on structural of the in the FP domain of Fbxo7 is to in PI31 (Fig. Fbxo7 and as a protein as to the PI31 in (Fig. we used in vitro binding to in vitro and Fbxo7 (Fig. that in binding PI31 (Fig. We also whether Fbxo7·Skp1 is to and whether this does Fbxo7 but not Fbxo7 (Fig. that Fbxo7 does indeed and that this interaction also the Fbxo7 on the binding for PI31 with a region of Fbxo7 required for homodimerization. We that as the in vitro Fbxo7 and of a for Skp1 in be PI31 and Fbxo7 data that the FP domains of PI31 and Fbxo7 be of in which have we whether a were into and and for Fbxo7 and PI31, both proteins were to be and a of Fbxo7 present in the (Fig. In Fbxo7 also within the with a in the in with the (Fig. The of PI31 an against the FP domain of PI31. on that the of PI31 and with a that the (Fig. We also that of Fbxo7 and PI31 in can be to (Fig. Fbxo7 is a subunit of an E3 ubiquitin ligase and in and with PI31, we whether Fbxo7 PI31 were with or against Fbxo7, as (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar), and were by for the presence of Fbxo7 and PI31. PI31 were by in Fbxo7 that does not the degradation of PI31 (Fig. how F-box proteins SCF substrates and other regulatory proteins through we on the Fbxo7 subunit of the SCFFbxo7. E3 ubiquitin ligase is reported to the degradation of HURP and (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar), and the Fbxo7 subunit acts as a of cyclin D-Cdk6 complexes (13Laman H. Funes J.M. Ye H. Henderson S. Galinanes-Garcia L. Hara E. Knowles P. McDonald N. Boshoff C. EMBO J. 2005; 24: 3104-3116Crossref PubMed Scopus (72) Google Scholar, H. Cell 2006; PubMed Scopus Google Scholar). identify further putative substrates and we structural, biochemical, and genetic approaches to study regions of Fbxo7 required for protein data the C-terminal as being required for binding to HURP and (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar). We that on Fbxo7 are required for its in interaction with within and the the C-terminal that Fbxo7 has a interaction with We have a domain of as the FP between the and F-box of with and we PI31, a of proteasome as with also an FP domain. proteins have not been PI31 and Fbxo7 not an FP domain in but also a conserved motif the proline-rich C Fbxo7 to be an of PI31, additional domains and which additional through regulatory and protein interaction The PI31 FP domain structure reveals an with structural to The domain as a both in and within of we that the PI31 FP domain through a and with the Fbxo7 FP domain through between their from S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) that PI31 with an of of a of for the FP domain that PI31 dimerization is through its FP domain. of that of the PI31 in present in complexes of which is with not the of PI31 is for its of immunoproteasome assembly is We also that Fbxo7 and PI31 via a on their FP domains and with an of In a of the proteins the not the that a of protein and to ubiquitin-proteasome whether Fbxo7 protein levels by the levels of PI31 were which against PI31 being a substrate for an SCFFbxo7 E3 this interaction be in or or Fbxo7 and PI31 as be other F-box as and have box domains that mediate and the structure of of domains has been recently X. S. Willems A. D. D. F. Sicheri F. Tyers M. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). are to the F-box to the FP domain of of has been shown to be required for its in and affect ubiquitin substrate binding (10Orlicky S. Tang X. Willems A. Tyers M. Sicheri F. Cell. 2003; 112: 243-256Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, X. S. Willems A. D. D. F. Sicheri F. Tyers M. Cell. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). FP domain-mediated dimerization of SCFFbxo7 also be required for E3 ligase by complex The between the PI31 binding on Fbxo7 and Fbxo7 homodimerization an that PI31 SCFFbxo7 by Fbxo7 a model we are study and have that the C terminus of Fbxo7 binding to its substrates (17Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-325602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Chang Y.F. Cheng C.M. Chang L.K. Jong Y.J. You C.Y. Biochem. Biophys. Res. Commun. 2006; 342: 1022-1026Crossref PubMed Scopus (54) Google Scholar), and the of PI31 to bind and proteasome has also been to the C terminus S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). this region is the conserved between the the Although its is the motif in PI31 a binding In PI31 has in its C act as binding for which protein interactions with proline-rich M. K. M. C. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Fbxo7, does not the the from a of Fbxo7 and PI31 their to as that the terminus of Fbxo7, have also been reported to bind to ubiquitin of the proteasome (16Hicke L. Schubert H.L. Hill C.P. Nat. Rev. Mol. Cell. Biol. 2005; 6: 610-621Crossref PubMed Scopus (639) Google Scholar), a link with PI31 as both proteins are of with the proteasome (19Chu-Ping M. Slaughter C.A. DeMartino G.N. Biochim. Biophys. Acta. 1992; 1119: 303-311Crossref PubMed Scopus (115) Google Scholar, 20Zaiss D.M. Standera S. Holzhutter H. Kloetzel P.M. Sijts A.J. FEBS Lett. 1999; 457: 333-338Crossref PubMed Scopus (77) Google Scholar, 21McCutchen-Maloney S.L. Matsuda K. Shimbara N. Binns D.D. Tanaka K. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 2000; 275: 18557-18565Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In we have regions required for interaction within Fbxo7 and a novel dimerization domain that is also present within the PI31. The link between Fbxo7 and PI31 both a structural and is further by the interaction between within both proteins ablate this and this can be in to the of their and the of their by the the We from and in and