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Molecular Cell, Vol.

11, 1445–1456, June, 2003, Copyright 2003 by Cell Press

Structure of a !-TrCP1-Skp1-!-Catenin Complex:
Destruction Motif Binding and Lysine Specificity
of the SCF!-TrCP1 Ubiquitin Ligase
Geng Wu,1,2 Guozhou Xu,1 cases governed by the phosphorylation or other post-
Brenda A. Schulman,1,4 Philip D. Jeffrey,1 translational modification of the substrate. E3s are a
J. Wade Harper,3 and Nikola P. Pavletich1,2,* diverse family of proteins and protein complexes, and
1
Cellular Biochemistry and Biophysics Program and they mediate ubiquitination in at least two distinct ways.
2
Howard Hughes Medical Institute HECT-type E3s first form an E3-ubiquitin thioester con-
Memorial Sloan-Kettering Cancer Center jugate and then transfer the ubiquitin to the substrate.
New York, New York 10021 RING-type E3s, which are characterized by the presence
3
Verna and Marrs McLean Department of a RING domain that binds the E2, do not form an E3-
of Biochemistry and Molecular Biology and ubiquitin conjugate, and they are thought to promote
Howard Hughes Medical Institute the ubiquitination of the substrate directly by the E2
Baylor College of Medicine (reviewed in Pickart, 2001).
Houston, Texas 77030 The SCF is a four-subunit RING-type E3, and it repre-
sents the largest family of E3s known to date (Deshaies,
1999). SCF family members regulate the cell division
Summary cycle, transcriptional pathways, and multiple aspects of
development, and they also play a central role in the
The SCF ubiquitin ligases catalyze protein ubiquitina- phosphorylation-mediated destruction of regulatory
tion in diverse cellular processes. SCFs bind substrates proteins. SCF complexes are composed of the scaffold
through the interchangeable F box protein subunit, with protein Cul1, the RING-domain protein Rbx1/Roc1, the
the !70 human F box proteins allowing the recognition adaptor protein Skp1, and an F box protein that binds
of a wide range of substrates. The F box protein the substrate. Rbx1 associates with Cul1 and the E2
!-TrCP1 recognizes the doubly phosphorylated (Skowyra et al., 1999), while Skp1 interacts simultane-
DpSGφXpS destruction motif, present in !-catenin ously with Cul1 and with the F box protein (Bai et al.,
and I"B, and directs the SCF!-TrCP1 to ubiquitinate these 1996; Feldman et al., 1997; Skowyra et al., 1997). F box
proteins at specific lysines. The 3.0 Å structure of a proteins constitute the largest known class of E3 speci-
!-TrCP1-Skp1-!-catenin complex reveals the basis of ficity components. Human and mouse genomes contain
substrate recognition by the !-TrCP1 WD40 domain. more than 70 F box proteins while C. elegans contains
The structure, together with the previous SCFSkp2 struc- 326 predicted F box proteins (Winston et al., 1999b;
Kipreos and Pagano, 2000; J.W.H and J. Jin, unpub-
ture, leads to the model of SCF catalyzing ubiquitina-
lished data). F box proteins interact with Skp1 through
tion by increasing the effective concentration of the
the !40 amino acid F box motif (Bai et al., 1996; Schul-
substrate lysine at the E2 active site. The model’s pre-
man et al., 2000) and with substrates through C-terminal
diction that the lysine-destruction motif spacing is a
protein-protein interaction domains, including WD40 re-
determinant of ubiquitination efficiency is confirmed
peats (Fbw subfamily) and leucine-rich repeats (LRRs;
by measuring ubiquitination rates of mutant !-catenin
Fbl subfamily) (Deshaies, 1999). In addition to this origi-
peptides, solidifying the model and also providing a
nal SCF family, there is at least one other family of SCF-
mechanistic basis for lysine selection.
like ubiquitin ligase complexes that are composed of
the Cul1-paralogue Cul2, Rbx1, the Skp1-like adaptor
Introduction protein ElonginC, and a SOCS box protein. SOCS box
proteins are structurally and functionally related to F
Ubiquitin-mediated proteolysis has a regulatory function box proteins (Stebbins et al., 1999; Schulman et al.,
in many diverse cellular processes (Hershko and Ciecha- 2000), and the SOCS box protein VHL has been shown
nover, 1998). The ubiquitination of a specific protein is to bind to the hypoxia-inducible transcription factor 1-"
carried by the ubiquitin-protein ligases (also known as (Hif1-") and promote its ubiquitination by the SCF-like
E3s), which act at the last step of a three-enzyme cas- ubiquitin ligase (Ivan and Kaelin, 2001).
cade involving ubiquitin-activating (E1) and ubiquitin- Much of our understanding of SCF function and the
conjugating enzymes (E2). The E1 activates the ubiquitin role of phosphorylation in protein turnover has come
in an ATP-dependent reaction and transfers it to the E2 from the analysis of the WD40 repeat containing F box
through the formation of a thioester bond between the protein #-TrCP1, which is conserved from C. elegans to
E2 active site cysteine and the ubiquitin C terminus. The humans. #-TrCP1 has been biochemically and geneti-
E3 binds both a cognate E2 and the substrate, and is cally demonstrated to control the stability of proteins
responsible for conferring substrate specificity to the involved in transcription, including #-catenin (Hart et al.,
ubiquitination reaction (reviewed in Pickart, 2001). The 1999; Latres et al., 1999) and I$B (Li and Verma, 2002).
binding of the E3 to the substrate protein is also the All known SCF#-TrCP1 substrates contain the DSGφXS
primary event regulating protein stability and is in many destruction motif (φ representing a hydrophobic and X
any amino acid). Phosphorylation of both serines of the
*Correspondence: nikola@xray2.mskcc.org
destruction motif is a prerequisite for #-TrCP1 binding,
4
Present address: Departments of Structural Biology and Tumor Cell linking phosphorylation-mediated signaling to protein
Biology, St. Jude Children’s Research Hospital, Memphis, Tennes- ubiquitination and destruction. In the case of I$B, which
see 38105. holds the transcription factor NF$B in an inactive cyto-

The #-TrCP1 linker domain consists ported by several mutagenesis studies.. It has been proposed that the SCF and other RING.. centered without significantly affecting its ability to bind either on the doubly phosphorylated destruction motif (resi- substrate or E2s (Joazeiro et al.6 Å eral aspects of the positioning model have been sup. and hydrophobic residues of the the Hif1" substrate. we have determined the 3. 2000). The Skp1-#-TrCP1-#-catenin complex has an elon- E3s catalyze ubiquitination through a mechanism that gated. 1999. position syndrome (Stebbins et al. These structures torus-like structure (named # propeller) that is character- revealed that individual E3 subunits and domains are istic of this fold (Wall et al. 2000) to a Hif1" substrate peptide (Stebbins et al. opposite from where the linker packs (Figures 1A tions in c-Cbl that affect the rigid linkage between the and 1B).. 2002. in part. 1999). Hon et al.. Muta- peller. 2002). that gets ubiquitinated by the SCF#-TrCP1 in vitro. #-TrCP1 fragment (residues 139 to 569) that lacks a 138 tion-associated genes (Polakis. 2003). and of the RING E3 c-Cbl boxy-terminal regions. destruction motif are recognized directly by contacts quently mutated in the von Hippel Lindau cancer-predis. Tumor-derived residue N-terminal portion of unknown function. of the LRR-con. #-TrCP1 consists of the substrate relative to the E2 active site (Schulman et the N-terminal F box domain (residues 139 to 186). cytokines induce the phosphorylation model that the SCF catalyzes ubiquitination by increas- of the two serine residues (Ser32 and Ser36) in the I$B ing the effective concentration of the substrate lysine destruction motif. 2000). The phospho- residues important for the relative arrangement of its " and # domains.. Min but shows considerable variation in its amino.. a al. leading to its stabilization. the structure !-Catenin Binding of yeast Skp1 bound to the WD40-containing F box As seen with other WD40 domain structures. 2002). Further sup- porting the positioning model of catalysis. 1995). Latres et al. and Cdc4 mutations 1B). The #-TrCP1-Skp1 complex was produced in insect phorylation. The structure....5–1. 1998. The #-TrCP1 # propel- connected through rigid structural couplings without ler is very similar to the canonical # propeller structure. The middle portion of the peptide has SCF#-TrCP1. The seven WD40 repeats form a bound to an E2 (Zheng et al. leads to the refined and the rest of the BC loops (Figure 1B). of NF$B (Winston et al. 1999. and the two ends have turn- of the human #-TrCP1-Skp1 complex bound to a 26 like conformations. Gen.. The taining F box protein Skp2 bound to Skp1 (Schulman et #-TrCP1 F box motif has the same overall three-helix al. aspartic acid. !-Catenin Complex Winston et al. 1995) and Tup1 (Sprague et al. and transcriptional activation of prolifera. 1999). together with mutagenesis and it can be superimposed on the WD40 domains of (Zheng et al. in contribute contacts to #-catenin. 2000)... is ordered in the structure. 2002).and car- et al. 2002). Wnt signals block #-catenin phos. 1999a. 2000. leading to its ubiquitination by at the E2 active site. from #-TrCP1. over 255 C" atoms. of the VHL-ElonginC-ElonginB complex bound cluster structure seen with Skp2 (Schulman et al. any flexible linkages and. 2002)... binds the top face of the # propeller. Mutation of a Skp1 residue important for #-TrCP1 F box in a manner similar to the Skp1-Skp2 the precise arrangement of the Skp1-Skp2 interface was complex (Schulman et al. Li and Verma. Two-thirds of the con- conjunction with measurements of in vitro ubiquitination tacts involve the A strands that line the central channel rates of mutant #-catenin peptides. This mutations in APC or in #-catenin. prevent human #-catenin substrate peptide (residues 19 to 44) #-catenin ubiquitination (Polakis. previously shown to have a BTB/POZ fold struc- affecting its ability to bind the substrate or the E2 (Zheng ture followed by two helices (H7 and H8). also helped eliminate the alternative G# (Wall et al. and also provides a mechanism SCF#-TrCP1.... the protein Cdc4. 2000) mechanism of acid/base catalysis by E3 residues.Molecular Cell 1446 plasmic complex. 2000). reported when this manuscript was first #-TrCP1 # propeller has a narrow channel running submitted.. with Skp1 and the #-catenin involves the recruitment and productive positioning of peptide located at opposite ends.. 1995). By contrast. the phosphorylation of Results and Discussion the destruction motif of #-catenin (Ser33 and Ser37) by the GSK3#-APC-Axin complex occurs in the absence Overall Structure of the !-TrCP1-Skp1- of Wnt signals (Hart et al. linker domain) linking the two (Figures 1A–1C). All seven WD40 repeats of #-TrCP1 residue #-catenin substrate peptide. Zheng et al. . with root-mean-square deviations (rmsd) of 1. This model emerged. VHL dues 32 to 37). scaffold was shown to inactivate the SCFSkp2 without Skp1. which are among the complex was crystallized bound to a 26 amino acid most common mutations in colon cancer. 1999a). hereafter boxSkp2 complex (Zheng et al. 1999). were found to be fre. The introduction of four " helices. translocation to cells by coexpressing human Skp1 with a human the nucleus. E2 and substrate binding domains abolished its function only an 11 residue segment (residues 30 to 40). and it interacts extensively with both of flexible linkers that eliminate the rigidity of the Cul1 the F box and with one face of the WD40 # propeller. shown to disrupt Skp1 function in yeast without abolish- The #-catenin peptide binds to one face of the # pro- ing Skp1-Skp2 binding (Schulman et al. C-terminal WD40-repeat domain (residues 253 to 545). 1999.. from the structures of the Cul1-Rbx1-Skp1-F and an "-helical domain (residues 187 to 252. 1999).. Of the 26 #-catenin residues in the crystals. with the channel being narrowest near the top face designed to affect the rigid coupling disrupted function (convention of Wall et al. respectively. The #-catenin peptide in vivo (Orlicky et al. and liberation for the selection of lysine(s). 2000..0 Å crystal structure an extended conformation. destruction by the proteasome. 2002).. also showed rigid coupling between the F through the middle of the torus-like structure (Figure box and substrate binding domain.. with the six residue To further elaborate on the mechanistic details of this destruction motif dipping into the central channel (Fig- model and to understand the substrate specificity of the ures 2A and 2B). In addition.. curved structure. which bind the ElonginC adaptor and serine. Yaron et al. binds the et al.

making intermolecular contacts in a mostly Tyr271 hydroxyl group. The side chains Ser325. at opposite sides of residues. Figures were prepared with the programs MOLSCRIPT (Kraulis. and it partially dips into the central channel. The seven blades of the WD40 domain are numbered from 1 to 7. and those mediating WD40 domain-linker interactions by purple crosses. Skp1 blue. The consensus destruction motif is shown below the alignment. and the backbone amide nium group of Arg285 (Figure 2A). with dashed lines indicating regions that are disordered in the crystal structure. Gly34. number of contacts. Dotted lines represent disordered regions. (C) Top panel: Sequence conservation and secondary structure elements of human #-TrCP1 (residues 139 to 569). Gly432. #-TrCP1 is colored red. Asp32. Residues identical in all four orthologs are highlighted in yellow. and #-catenin yellow. and electrostatic interactions with the guanidi- The phosphate group of pSer33 makes the largest nium group of Arg431 (Figures 2A and 2B). Essar. 1991). Ser309. GL_RENDER. creating a hydrogen bond net- buried environment. The #-catenin residues that have clear electron density in the crystal structure are underlined. The phosphate group of pSer37 contacts fewer and pSer37 bind sites at the rim. Bottom panel: Sequence conservation of the human #-catenin peptide (residues 19 to 44) used in crystallization. which . (B) The phosphorylated #-catenin peptide binds to the top face of the WD40 domain of #-TrCP1. forming hydrogen bonds with the hydroxyl the channel. and six residue destruction motif (Figure 2A). #-TrCP1 residues that interact with Skp1 are indicated by blue dots and with #-catenin by red triangles. personal communication). #-TrCP1-interacting residues of #-catenin are indicated by red triangles. The phosphate groups of pSer33 work. with φ representing a hydrophobic residue and X any residue. Structure of the Skp1-#-TrCP1-Phosphorylated #-Catenin Peptide Complex (A) Overall view of the complex.#-TrCP1-Skp1-#-Catenin Structure 1447 Figure 1. In addition. The secondary structure elements for Skp1 and for the F box and linker domains of #-TrCP1 are labeled. Nearly all of the #-TrCP1 contacts are made by the side chain hydroxyl groups of Tyr271. the back- and carbonyl groups of His36 insert the farthest into bone amide of pSer33 also hydrogen bonds to the the channel. and POVRAY (L. #-TrCP1 residues that mediate F box-linker interactions are indicated by green rectangles. forming hydrogen bonds with the Among the buried #-catenin residues. and the strands within each blade are named A to D according to the convention of Wall et al. (1995). group of Ser448 and the backbone amide group of posed. and their contacts are partially solvent ex. and Ile35. and electrostatic interactions with the guanidi- of Asp32.

be. (1996) noted that three residues tween the Leu31 backbone carbonyl group of #-catenin of the WD40 motif are repeatedly involved in interactions and the Arg521 guanidinium group of #-TrCP1. and the #-catenin peptide is shown in orange. start of the A strand (A-1 position). (B) Surface representation of the top face of the #-TrCP1 WD40 domain with the #-catenin peptide bound. makes the most Three Positions of the WD40 Motif Make extensive contacts (Figure 2A). two positions are the residue immediately prior to the makes van der Waals contacts with Ala434. The diately after the B strand (B&1 position). a turn that packs only loosely on the surface of the These similarities highlight the central channel as a #-propeller. and the residue imme- and the aliphatic portion of the Arg474 side chain. which is in the only variable position four repeats. I$B" and I$B"-ubiquitin conjugates were detected by immunoblotting with anti-I$B" antibodies. Its side chain makes a the Majority of Contacts charge-stabilized hydrogen bond with Arg474 and an. also an invariant destruction motif WD40 repeat motif. Leu 472. The #-TrCP1 WD40 domain is shown in red. A strand (the A2 position. each used by side chain of His36. Gaudet et al. Control reactions (lane 1) contained unprogrammed reticulocyte extracts. binding of the transducin Gt#' to phosducin. The other whose hydrophobic nature is conserved in the motif. is the second residue of the ment with little space for a nonglycine residue. with only one contact. packs with Leu311 and Leu351 in an environ. channel. Figures 1B and 1C).Molecular Cell 1448 Figure 2. calculated in the absence of #-catenin with the program GRASP. (C) Mutational analysis of #-TrCP1 residues that interact with the destruction motif using an in vitro I$B" ubiquitination assay. The surface is colored according to the electrostatic potential (%20 kT to &20 kT). and B&1 positions of the #-TrCP1 repeats. but its backbone amide and carbonyl groups binding is similar to what has been observed in the make a pair of hydrogen bonds with the side chain of Asn394. through side chain . is an invariant destruction motif residue. Residues 38 to 40 following the motif form A-1. The majority (14/15) of the #-TrCP1 residues that contact other hydrogen bond with the hydroxyl group of Tyr488 #-catenin are located at one of three positions of the of #-TrCP1. by six of the seven repeats. used residue. made in with phosducin. of the motif. A2. structural scaffold that can adapt. with its side chains in pink. Gly34. Ile35. Hydrogen bonds are represented by white dotted lines. The ubiquitination assays (upper panel) were performed using a panel of #-TrCP1 mutants (lanes 2–10) produced by in vitro translation in the presence of 35S- methionine (lower panel). and these are the same residues as the this region. which has The two residues preceding the destruction motif a small helical domain that binds on the Gt# propeller point away from #-TrCP1. The Doubly Phosphorylated #-Catenin Peptide Binds to the Top Face of the #-TrCP1 WD40 Domain (A) Close-up view of the interface between the #-TrCP1 WD40 domain and the doubly phosphorylated #-catenin peptide. The most common position. with its side chains in yellow. points outward and has no interactions with The use of three positions of the WD40 repeat for #-TrCP1.

These Cdc4 specificity. On the basis of The #-TrCP1 F box binds Skp1 through a bipartite sequence and structure relationships from published . they adopt different and in the superimposition of the two SCFs roughly half structures as they pack with the different linker struc. One face of the F box interacts with the H5 and sequence over short polypeptide stretches. which makes fewer contacts. in #-catenin peptide point toward Rbx1 (Figure 4). 22 and 30 cases. while the opposite face interacts with the pSer37 bind to very similar sites on repeats 2 and 5. of SCF#-TrCP1 activity in this assay. and H7) of the linker pack Motif Recognition into a globular domain that is anchored tightly on the F To further explore #-TrCP1-destruction motif recogni. domains that are highly analogous to those made by the tion of #-catenin mutations identified in colorectal. pSer37. interacting with the and Skp2 linker structures.8 Å rmsd for to the steric hindrance by the #-catenin polypeptide 102 C" atoms). and. the serine contacts.. constructed a model of the SCF-like VHL-ElonginB- Figure 3A). While the H1 and H2 helices the top face of the WD40 domain faces the E2 binding align well. and both the N and C termini of the from the H2 helix by !15( (Figure 3A). relative to Skp1. endometrial. of the WD40 domain structure overlaps the LRR domain tures that follow the F boxes (Figure 3B). in the case of Skp2. thus reinforces the notion that the rigidity of the linkage viewed in Polakis. shifted together by !2. ElonginC-Cul2-Rbx1 (VBCCR) complex. and tested the ability of single. linker helices make contacts to the F box and WD40 plays a smaller role. resulting in complete loss www. 7. the H3 helix of #-TrCP1 is tilted further away site on Rbx1. In this SCF#-TrCP1 model. While these residues match the F box consensus the position of the LRR domain in the SCFSkp2 structure. Mutation of and hydrogen bond networks involving conserved resi- Arg474 or Tyr488. Gly 34. Asp32. makes additional contacts with the F box (Figure 3D). 2003) with the Skp1-Skp2 a coil structure that interacts with the LRR-like linker. pSer33.org/cgi/content/full/11/6/1445/DC1). pSer37. well in both #-TrCP1 and Skp2. The additional con. 2000). The residues that tion. the phosphate groups of pSer33 and helix of Skp1.molecule. which contains a 5 residue insertion in the positions of the substrate binding domains. mutation of Arg431 and Tyr271 resulted linker forms a three-helix globular domain (Orlicky et al. The #-TrCP1-Skp1 complex tocellular. bound SCF by simply superimposing the Skp1-F box helix cluster structure (H1. The rest of the Skp1 structure is and B&1 positions) are not possible for pSer37 due essentially identical in the two complexes (0. This is also reflected in the distribu. 23. dues (Figure 1C and Supplemental Figure S1 at http:// nine had the biggest effect. 2002).. Linker between the F Box and WD40 Domain Mutational Analysis of !-TrCP1-Destruction The four helices (H4. both of which contact Asp32. H8 helix. Asp32. but there are several differences (1. respectively. to ala. In a recent survey (Polakis. 1999a) (Figure 2C). In #-TrCP1. On the WD40 side. Model of the SCF!-TrCP1-!-Catenin Complex The structure of the Skp1-#-TrCP1-#-catenin complex F Box and Its Interface with Skp1 makes it possible to construct a model of a substrate- The #-TrCP1 F box contains the same overall three.. which pack with the F box A2 and B&1 positions of repeats 3 and 4 (Orlicky et al. second difference involves the N terminus of the To further explore the significance of this similarity #-TrCP1 F box. A similar overlap of substrate binding domains they form an " helix that continues as the first helix of has also been reported for the superposition of the yeast the helical linker domain.5 Å complexes (Zheng et al. but the positions of the (Ser309 and Gly432) and B&1 (Ser325 and Ser448) posi. in significant loss of activity. and ovarian cancers (re. The part. Two of the Cdc4 linker helices have positions and structure-based conclusion that Asp32. Ser 33. with Skp1.#-TrCP1-Skp1-#-Catenin Structure 1449 changes. to the six C-terminal F box residues (residues 187 position of the WD40 domain is remarkably similar to to 192). and H6 helices of the BTB/POZ core and with the H7 Remarkably. and 1 through van der Waals contacts system (Winston et al. and the linker H7 helix of the destruction motif. The movement of the F box is associated backbone preceding pSer37. the linker H5 and H7 helices pack ated polyubiquitination of I$B" in an in vitro translation with blades 6. This is due. orientations. #-TrCP1 F box and of the Skp1 H7 and H8 helices are tions of these repeats (Figure 2A). #-TrCP1 H4 and H7 helices. H6. to recognizing diverse secondary structure interface. while Ile35. A structure.. we (Figures 3A and 3B) that forms an additional " helix (H0. and they share contacts similar to the A-1 in the Skp1-Skp2 complex. 2003). In the structure of the yeast Skp1-Cdc4 complex. In the recently reported with the #-TrCP1 H0 helix wedging into the expanded structure of the yeast Skp1-Cdc4 complex bound to space between the BTB/POZ core and one face of the a phosphopeptide selected from a library. gastric. box and WD40 domains (Figure 1A). and H3) seen with the F portions of the Skp1-#-TrCP1-#-catenin and the SCFSkp2 box of Skp2.. site #-TrCP1 mutants to promote the SCF#-TrCP1-medi. between the F box and the substrate binding domain is 2000). while there were only two mutations reported for Ile35. rmsd for 37/42 C" atoms). (Figure 4). This plasticity in the F box-Skp1 inter- phosphothreonine binds the Cdc4 WD40 domain in a face is likely related to the entirely different #-TrCP1 location similar to that of pSer37. that are very similar to the and Gly34 are critical for #-TrCP1-#-catenin binding and H4 and H7 helices of the #-TrCP1 linker. H2. and Ser37 were found important for SCF function (Schulman et al. mutated in 36. whereas in Skp2 they adopt Skp1-Cdc4 (Orlicky et al. These findings support the 2003). Among the phospho. the single F box (Figure 3C). H5. hepa. 2000). This binding mode is very similar to that seen respectively.0 Å away from the BTB/POZ core tacts that pSer33 makes to the neighboring repeat 1 (A2 of Skp1 (Figure 3C).. and Ile36 residues the C terminus of the F box motif. we mutated eight of the #-TrCP1 residues that form the first turn of the linker H4 helix correspond to contact the pSer33.

as c-Cbl also uses a domain out- binding domain of VHL (termed # domain) points toward side its RING to bind the E2. 2000). The the E2-Rbx1 interface (Figure 4). a 45( distance from the Hif1" peptide to Rbx1 is !25 Å longer uncertainty in the orientation of the E2 could result compared to the SCF#-TrCP1-#-catenin peptide model in )20 Å errors in the position of the E2 active site (Figure 4). 1999. Zheng E2 model. with the N terminus of the Hif1" peptide in sight is not apparent in the SCF.. the linker in green. the E2 active site cysteine would end up et al. and Skp1 (cyan) that are involved in intermolecular or interdomain contacts are also shown. The substrate active site. position of the E2 active site.. 2002) complexes (Figure 4). Motif Spacing plex and on mutagenesis indicating that the Rbx1 RING The similar positions of the three substrate binding do- domain has an E2 binding site similar to that of c-Cbl’s mains supports the current model that the SCF and other RING domain (Zheng et al. The #-TrCP1 and Skp2 secondary structure elements and residue numbers are labeled above and below their sequences. respectively. (D) Close-up view of the interface between #-TrCP1 and Skp1. Side chains of the #-TrCP1 F box (pink). and their comparison to the F box motif consensus. and those in Skp2 are indicated by green crosses. RING E3s promote ubiquitin transfer by positioning the . may have a significant error in the position of the E2 tide (Min et al. Identical residues between #-TrCP1 and Skp2 are highlighted in yellow. The SCF-E2 model Skp1-F boxSkp2 and VHL-ElonginB-ElonginC-Hif1" pep. which is !20 Å away from although it does not extend as far toward Rbx1. The #-TrCP1 F box is shown in red. The structures were superimposed by aligning the BTB/POZ core of Skp1 in the two structures. cysteine relative to the SCF. !50 Å away from the structured N.or C-terminal ends served portions of Skp1 and ElonginC from Cul1-Rbx1. we superimposed the structurally con. Skp1-interacting residues in #-TrCP1 are indicated by blue dots. (C) The #-TrCP1 F box and Skp1 H7-H8 helices are shifted en bloc away from the Skp1 BTB/POZ core compared to the structure of Skp1- Skp2. linker (light green). and a counterpart to this Rbx1. Pivoting about the E2-Rbx1 of Rbx1. 2000. Secondary structure elements of the #-TrCP1 F box are labeled. For example.Molecular Cell 1450 Figure 3. and Skp1 in purple. Schulman et al. Hydrogen bonds are represented by white dotted lines. The position of the VHL # domain is again very interface of the model could significantly affect the final similar to the #-TrCP1 WD40 and Skp2 LRR domains. but a model of the SCFSkp2 bound to an E2 has been Ubiquitination Rate Depends on Lysine-Destruction proposed based on the structure of the c-Cbl-E2 com. Based on this SCF.. work (Stebbins et al... of the #-catenin peptide (Figure 4). The #-TrCP1-Skp1 Interface Is Similar to that in the Skp1-Skp2 Complex (A) Superimposition of the #-TrCP1 (red) and Skp2 (green) F boxes. The structure of the E2-Rbx1 complex is unknown. showing intermolecular contacts. however. (B) Structure-based sequence alignment of the #-TrCP1 and Skp2 F box sequences. 2000).

. together with the conservation of (Zheng et al. One prediction of this position. The E2 active site cysteine. 2002).. 2002). Models of the SCFSkp2 (Top). The model of the VBC-CR complex (Table 1). #-catenin in vivo. suggest that Lys19 is a ubiquitination site of contains a model of Cul2. 2002). SCF#-TrCP1 (Middle). all purified to !90% homogene- ity. As shown in Figure 5A.. two long-range structural effects because the N-terminal adjacent lysines (Lys21 and Lys22) have been shown 133 amino acids that encompass this region have been to be necessary and sufficient for ubiquitination and shown. We decreased the spacing by deleting 4 (residues 21 to 24. in the crystallized peptide but are disordered in the ciency of SCF#-TrCP1-mediated ubiquitination. In order to be able to accu- rately measure the ubiquitination of the #-catenin lysine independent of the ubiquitination of lysine(s) on ubiqui- tin.#-TrCP1-Skp1-#-Catenin Structure 1451 Table 1. The #-cate. 1997). structure.. face. These findings. and a model of the Cul2-ElonginC inter. all of the I$B" and #-catenin orthologs and paralogues also contain a lysine located 9 to 14 residues upstream of the destruction motif (Table 1). The deleted residues and ing model is that the location of the ubiquitinated lysine the residue where the linkers were inserted are present relative to the destruction motif is important for the effi. Replacing SCF#-TrCP1 with the SCFSkp2-Cks1 complex.. Distance to Nearest Lysine Upstream of Destruction Motif Substrate Residues I$B" (human) 9 and 10 I$B# ( human) 9 I$B* ( human) 11 Cactus (fly I$B) 10 and 12 #-catenin (human) 13 Armadillo (fly #-catenin) 14 Bar-1 (worm #-catenin) 10 Plakoglobin (human) 11 lysine (Lys19) 13 residues upstream of the destruction motif (Figure 1C).2 mM).. Baldi et al. and SCF#-TrCP1. did not produce any detectable ubiquitinated #-catenin. motif was altered in 4 residue steps. When we use the wild-type ubiquitin instead of the R7 mutant. the #-catenin peptide is ubiquitinated in an SCF#-TrCP1-dependent manner to an overall level comparable to the I$B" substrate peptide (compare lanes 3 and 6). is shown in space-filling representation and is colored cyan. The mutations are also unlikely to have mapped. at the same site (Figure 5C). In addition. We increased the spacing by inserting presented to the E2 active site (Schulman et al. Inspection of the compared to ubiquitin and peptide substrates (0. 1996). wt%8) substrate in a manner such that the lysine is optimally starting at Ala21. but in the case of the I$B" substrate. wt%4 peptide in Figure 5C) or 8 residues (residues 21 to 28.. based on the structure of the VHL-ElonginB-ElonginC-Hif1" We next tested a series of mutant #-catenin peptides peptide complex (Min et al. 2001). 2002) and on the RING-type E3 c-Cbl bound to the this lysine among #-catenin orthologs and paralogues UbcH7 E2 (Zheng et al.. The ubiquitination assays were per- These two I$B" lsyines are located 10 and 9 residues formed using a catalytic amount of SCF#-TrCP1 (1. 2000. to be unstructured degradation (Scherer et al. which would be attached to the ubiquitin C terminus through a thioester where the spacing between Lys19 and the destruction bond. by proteolytic digestion. demonstrating that our assay conditions maintain the substrate specificity of the SCF#-TrCP1 (Figure 5A.. we used the R7 ubiquitin mutant that lacks lysines and thus does not form polyubiquitin chains (Zhou et al. E2 (UbcH5). 1995. We thus tested whether a #-catenin peptide con- taining Lys19 and the destruction motif can be ubiquiti- nated in an in vitro reaction reconstituted with E1. compare lanes 3 and 8). 2000). a Gly-Ser linker of either 4 (wt&4) or 8 (wt&8) residues Zheng et al. indicating that they are not involved in nin lysine(s) that gets ubiquitinated has not yet been #-TrCP1 binding. and the that can be seen by either Coomasie staining or by SCF-like VBC-CR Complex (Bottom) Bound to E2 immunoblotting with anti-#-catenin antibodies (Figure The E2 was docked based on the composite structure of the SCFSkp2 5B). which was previously shown to ubiquitinate the p27Kip1 substrate in vitro (Zheng et al.. (Huber et al. human #-catenin sequence showed that it contains a The mutant and wild-type #-catenin peptides and the . 2000.9 +M) upstream of the destruction motif. the reaction yields the expected high- molecular weight polyubiquitinated #-catenin peptide Figure 4.

and doubly ubiquitinated peptides are indicated. The gels were visualized either by Coomassie staining (left panel) or by Western blot (right panel) using anti-phospho-#-catenin antibodies (Cell Signaling).Molecular Cell 1452 Figure 5. wt&4. the in vitro system yields high molecular weight polyubiquitinated products. except for the #-catenin concentration. (D) Ubiquitination of the wt%8. the SCF#-TrCP1 monoubiquitinates the doubly phosphorylated #-catenin (lane 3) and I$B" (lane 6) peptides at comparable rates. E2. and E3 enzymes. . When the R7 mutant of ubiquitin is used. The reaction is dependent on Lys19 in the #-catenin peptide (lane 5) and is specific for #-TrCP1 because Cul1-Rbx1 (lane 7) or SCFSkp2-Cks1 (lane 8) cannot substitute for SCF#-TrCP1. which is 50-fold less. wt%4. wt&4. (E) Plot of the ubiquitinated peptide/total ubiquitin ratio as a function of time. Bands corresponding to free ubiquitin. the ubiquitinated lysines in pink. and each set was repeated four times. and wt&8 #-catenin peptides. wild-type. ubiquitinated peptides. The dissociation constants (KD) and standard deviations of curve fitting are indicated in the inset. wt&8 #-catenin peptides. The average and standard deviations of four experiments for each peptide are plotted. The Rate of Ubiquitination by the SCF#-TrCP1 Is Dependent on the Spacing between the Ubiquitination-Site Lysine and the Destruction Motif (A) The reconstitution of an in vitro ubiquitination system with purified E1. (B) When wild-type ubiquitin is used. and the residues inserted in the wt&4 and wt&8 mutant #-catenin peptides in green. The destruction motif is highlighted in yellow. The SDS-PAGE gel was visualized by Coomassie staining. the Cul1 C-terminal fragment also gets ubiquitinated (Ub-Cul1-CTD). (F) Binding of the #-TrCP1-Skp1 complex to the monoubiquitinated wt%4. (C) Sequences of the wild-type and mutant #-catenin and I$B" peptides used in the in vitro ubiquitination assay. Substrates and products were visualized by Coomassie staining following SDS-PAGE. In this system. wild-type. The reaction conditions are identical to those in (A). and of the wild-type I$B" peptide at different time points. All six peptides were assayed at the same time.

We compared the wt%4 peptide. but do not eliminate it. closely reach the E2. that is ordered in the crystals (Tyr30). estab. as the wt%4 peptide can reach only !32 Å in a to !2-fold. we used a random polymer model of 7 units clusion that the insertions and deletions in the #-catenin (7-mer) corresponding to the number of residues be- peptide would not affect the association of the peptides tween the lysine of wt%4 and the first #-catenin residue with #-TrCP1. but consistently. 1975. approximately 3-fold lower apparent rate. The length distri- We found that changing the Lys19-destruction motif bution of an unstructured polypeptide can be described spacing had a significant effect on the ubiquitination by an average length and a standard deviation related rate (Figures 5D and 5E).. higher than that Creighton. where the lysine-destruction motif served differences in the ubiquitination rates of the mu- spacings of 9 and 10 residues is comparable to the 9 tant peptides are consistent with the effective concen- residue spacing of the wt%4 mutant. and wt&8 mutant #-catenin pep. such as the alignment of the lysine side discussed earlier. the ubiquitination rate of the wt%4 distributions of related peptides (Haas et al. with accuracy. The average end-to-end distance precisely. the SCF#-TrCP1 is most efficient when a lysine is present ation. The KD values of that the 19-mer would sample this distance is lower by the Skp1-#-TrCP1 bound to monoubiquitinated wild. struction motif and the ubiquitin-acceptor lysine residue Our experimental data showing that ubiquitination by as a parameter that affects the rate of substrate ubiquiti. a parameter the wt%8 peptide. which would likely be too short to likely unique to a particular F box protein. For the wt%4 To experimentally confirm the structure-based con. we used peptides that have been monoubiqui. ranging from 480 to 566 nM time that the lysine spends near the E2 active site. According to this effective If the lysine-destruction box spacing is smaller than concentration model. nated wt%8 produced at the longer reaction times. observe on varying the lysine-destruction motif dis- These data establish the spacing between the de. a factor of 2.#-TrCP1-Skp1-#-Catenin Structure 1453 wild-type I$B" peptide were assayed at the same time. which was ubiquitinated at an product is monoubiquitinated. of Ubiquitination Although we cannot calculate the average spatial The finding that 4 residue changes in the lysine-destruc. also is a slightly tration model. statis- the peptides than to that of wt%8 (Figures 5D and 5E). Similarly.. wt&4. instead was identical in all repetitions.5 (see Supplemental Figure S3). ate between those of the wt&8 and wt peptides. for the 7-mer was 2. we used a native gel electrophoretic mo. consistent with the magnitude of the rate effects we ubiquitinated product to perform this experiment. peptide. model predicts that it will not be ubiquitinated. we can put an upper tion motif spacing affect the rate of ubiquitination up limit. chain for nucleophilic attack.molecule. 4. The it is not possible to ab initio calculate these parameters wt&8 peptide was the second poorest substrate. which better substrate than wt #-catenin (compare the 5. and the or. 1993 and poorest substrate. this matches the spatial distance between the substrate’s could be due to a fraction of the peptide population . side chain. but we presume it (Zheng et al. references therein). Assuming type and wt%4. allowing the labeling with distance from #-TrCP1 to the E2 active site matches 32 P (see Supplemental Figure S2 at http://www. In the case in a 5 point time course (Figure 5D). especially as the amino acid sequence though its ubiquitination rate was closer to the rest of can significantly affect the distribution. However. of having a single. The wt%8 peptide was the to how broad the distribution is (Creighton. the probability org/cgi/content/full/11/6/1445/DC1). and 20 min time points. SCF binding motif and its lysine residue.35 units for the 19-mer. had the highest ubiquitination efficiency in our assay. It is note. Although strate binding site and the E2 active site. have been shown to agree with experimental data quali- lishing a trend of decreasing rates with increasing spac. further supporting the positioning model. In order to quantitate mental Figure S3 at http://www. Surprisingly. the segment between the ments was repeated four times (Figure 5E). well-defined length. The set of experi. a specific SCF will catalyze ubiqui. This is shorter than the !50 Å #-TrCP1-E2 tive concentration of the lysine at the E2 active site distance in the SCF#-TrCP1-E2 model. that the reaction rate is proportional to the fraction of tides are indistinguishable. tance. in Figures 5D and 5E). Except for very short polypeptides.molecule. compared to tinated with an R7 ubiquitin tagged with a cAMP-depen. with only trace amounts of ubiquiti. the tination maximally when the distance between its sub. of the #-catenin substrate. the average length of the optimal 7-mer. 2002) and not through a more precise is within the uncertainty in the E2 active site position mechanism. suggests that the fully extended conformation of its backbone and lysine positioning effect operates through increasing the effec. lengths of these residue spacings. 9 to 13 residues upstream from the destruction motif of the substrate indicates that this is the position that the Implications for SCF-Mediated Catalysis SCF#-TrCP1 maximally concentrates at the E2 active site. the with standard deviations of 68 to 107 nM (Figure 5F). peptide was slightly. which assumes each monomer unit is free to rotate.64 monomer units. what would minimally be required to reach the E2. Setting the condition that the dent-protein kinase (PKA) site. 1993). predictions of the effective concentration model are thus We could not obtain adequate amounts of the wt%8 mono. we used bility shift assay to measure the binding of the peptides a model of 19 units for the wt&8 peptide (see Supple- to the Skp1-#-TrCP1 complex. tatively. shows a low level of ubiquitination. al. of the wild-type peptide (Figures 5D and 5E). 10. and can be used to compare relative length ing. destruction motif and the lysine is unstructured and as dering of the peptides by their ubiquitination efficiency such it would sample a distribution of lengths.org/cgi/content/ binding and calculate the dissociation constants (KD) full/11/6/1445/DC1). when the majority of the I$B" and the wt&8 peptide. tical approaches based on random polymer theory. We applied polymer theory to see whether the ob- worthy that I$B". The wt&4 peptide was ubiquitinated at a rate intermedi.

95 3.0 (43. R factor .025–2. 0F. has been shown to block the the F1 beamline (.4 99.i!/E. Statistics from the Crystallographic Analysis Data Set Native Hg .58 – 1. and following al.2 3. The complex was tion of transiently overexpressed #-catenin (Aberle et first purified by glutathione affinity chromatography.4 20–3.895 Unique reflections 17.013 Rsym .9919 Resolution (Å) 2. trated to !15 mg/ml by ultrafiltration in 10 mM BTP.91 Mean FOM 0.94 – 0.014 Angles (() 1.I % Ih|//h/i Ih.2 Hg .. and this has been shown of reservoir solution containing 16%–20% PEG 4000.2) 6. 320 mM so- dium citrate. .5 Å. /||Fobs| % |Fcalc||//|Fobs|.10 Rcullis 0. Phasing power . with a .95 Reflections (|F| ! 0-) (3. It is thus conceivable that this is a second ubiqui. the two proteins in insect cells from separate baculoviruses.78 0.24 1.Molecular Cell 1454 Table 2.6 20–3.649 Side chain bond 2..5 (28.7) MAD Analysis Resolution 20–3. Rmsd. The human #-TrCP1-Skp1 complex was produced by coexpressing tholog. as well as by ubiquitination being one The purified #-TrCP1-Skp1 complex (15 mg/ml) and #-catenin pep- of many posttranslational modifications that occur at tide (5 mg/ml) were mixed at a 1:2 molar ratio. the distance criteria. #-catenin.950 Å) of the Cornell High Energy Synchrotron ubiquitination of the p53 tumor suppressor and the E2F-1 Source (MacCHESS) using crystals flash-frozen in crystallization ..i for the intensity (I ) of i observations of reflection h. and full-length Skp1 with two inter- but not of the individual lysines. and they that a lysine C-terminal to the destruction motif could also provide a mechanistic basis for the selection of also be ubiquitinated efficiently as long as it matches lysine(s) by the SCF. The #-TrCP1-Skp1 complex was then concen- lysines. sampling the vicinity of the E2 through random diffusion. complex in the asymmetric unit.522 15.528 Data coverage (%) 99. Interestingly.3 Wavelength (Å) 0.950 Å) 16. al- Protein Expression and Purification though this lysine is absent in the worm #-catenin or. And acetylation. In the SCF#-TrCP1-#-catenin-E2 model.793 (1201) Total atoms 4340 R factor (%) (3. The importance of ubiquitination-site specificity in regulation is reinforced by the reversibility Crystallization and Data Collection of ubiquitination.81 Anomalous Rcullis – 0.472 69. I$B" can be modified complex were grown at room temperature by the hanging drop by the ubiquitin-like protein SUMO on the same residues vapor diffusion method by mixing the complex with an equal volume that undergo ubiquitination.025–2. where 0F. 82.89 0.2 Observations 52.9500 1. 200 mM NaCl. The doubly phosphorylated mechanism for the selection of ubiquitin-acceptor ly. where Fobs and Fcalc are the observed and calculated structure factors. Crystals of the ternary lysine side chains.950 Å) 4.6 (51.4 20–3. Rcullis is the mean residual lack of closure error divided by the dispersive or anomalous difference.4 Phasing power 0. sines by the SCF.950 Å) 23.855 B factor (Å2) Main chain bond 0.0-2. which is another com. transcription factor (Li and Verma.1) Rmsd Bonds (Å) 0. c . Resi- tination site of #-catenin in vivo.0) 5. both the N and Our findings thus solidify the model that the SCF cata- C termini of the #-catenin peptide point toward the E2.3) Rfree (%) (3. This suggests tration of the lysine at the active site of the E2. The native data set was collected at mon lysine modification. 1997). by cation exchange and gel filtration degradation of endogenous #-catenin requires these chromatography.94 0. reduced the ubiquitina.393 85.0 (43. lyzes ubiquitination by increasing the effective concen- and they are roughly equidistant from it. 111. and contain one terro et al. #-catenin and I$B" wild-type and mutant peptides were synthesized chemically and purified by reversed phase chromatography.1 Hg .0080 0.. although it was not determined whether the cleavage of GST by thrombin.7 Rsym (%) (3. nal deletions of residues 38 to 43 and 71 to 82 (Schulman et al. 2000) was expressed as an untagged protein. 2002.0090 1. /h/i |Ih.8 (35.8).628 15. 6.48 Refinement Statistics Resolution range (Å) 20. transferase (GST) fusion protein.025–2.3 99. bas et al.950 Å) 28. This is supported by dues 139 to 569 of #-TrCP1 were expressed as a glutathione S the observation that mutation of both Lys19 and Lys49. Martinez-Bal- as part of the dynamic binding equilibrium. The effective concentration model also provides a 5 mM dithiothreitol (DTT) (pH 6. Rfree . 1998). 0.512 15. The crystals form in space to protect I$B" from ubiquitination and destruction (Des- group P31.025–2.6 Å. For example. root-mean-square deviations from ideal geometry and variations in the B factor of bonded atoms. R factor calculated using 5% of the reflection data chosen randomly and omitted from the start of refinement.2 3. and 100 mM BTP (pH . respectively.8). b . 2000).9) 5. and plakoglobin all have a lysine located 9 residues Experimental Procedures C-terminal to the last ordered #-catenin residue. armadillo.9 99.9 (28.i! is the rms heavy atom structure factor and E is the residual lack of closure error.111 66.

Accepted: April 25. Biol. homogeneity. Brown.9 +M). The amounts of ubiquiti. F. Lassot. 1997). A.M.. J. mixtures were separated by SDS-PAGE and I$B" detected by West. Kuszewski. yeast.. (1996). J.. D 50. G. and Deshaies. 209–219. P. Adams. 1991) and was improved by several cycles of manual rebuilding and refinement with the pro..S.J. The wild.D. The R7 mutant of ubiquitin was tagged with an N-terminal cAMP. Crystal structure at of 10 +l. Sen.J. 1807– to as PKA-Ub-R7).#-TrCP1-Skp1-#-Catenin Structure 1455 buffer supplemented with 20% ethylene glycol at %170(C (Table 2). R. (1997). Harper. It was expressed in E. 83 to 84. Pannu. models consisting of Skp1 (Schulman et al.. 1994). and visualized by phosphorimager analysis. C. N. (1999). and Kemler. Stappert.9 +M). Bohm.M. 1994).. 2003 under code 1P22. p27Kip1 in vitro (Zheng et al. A. (1975)... P.2 mM thimerosal for 1. Human UbcH5. R. U. (1996). C. K. the Dewitt Wallace residues 30 to 40 of #-catenin.Z. was analyzed by SDS-polyacrylamide gel electrophoresis. 16. phosphorylated with IKK (Winston et al. phosphorylated #-catenin and regulates its activity in the cell. D 54.. coli and was purified by 1811. and standard deviation were calculated and plotted with Microsoft and Polakis. indicated. 2000) and the G# WD40 domain (Sondek et al... This work was supported by #-TrCP1. The data were fitted into the quadratic equation of the lar replacement with the program AmoRe (CCP4. Franzoso. G. 271. We thank the staff of the Cornell High Energy Synchrotron Source grams CNS (Brunger et al.. Purified E1 (670 nM). PKA-Ub-R7 by scaling up the reaction described above. Cell 2. (1998).0). Rubinfeld. Proteins: structures and molecular properties C-terminal domains of Cul1 (NTD and CTD) and Rbx1 (Zheng et al.. Benarous. (1999). The ubiquitin system.S. Freeman and Company). Proc. and 10 mM magnesium chloride. and were incubated at room temperature for 30 min or as 2. H. Hart.T. Sci.. Residues 218 to 226 and 546 to 569 Foundation. supplemented with 10 mM ATP and 20 mM MgCl2 in a total volume Gaudet. The MacChess and of the National Synchrotron Light Source X9A beam- refined model contains residues 139 to 217 and 227 to 545 of lines for help with data collection. Acad.. M. pp.6 +M). Acta Crystallogr. quitin proteolysis machinary through a novel motif. The F-box protein #-TrCP associates with Excel. Annu. Cul1 Feldman. Biol. 9 +l of reticulocyte lysate containing in vitro translated Elledge. Bauer. Rodriguez. L. and gel filtration chromatography. Natl. 32P-PKA-Ub-R7- DENZO and SCALEPACK (Otwinowski and Minor. USA 72. (New York: W. the Howard Hughes Medical Institute. (1993).W. ubiquitin-mediated pro- teolysis of I$B". The free and (MAD) using a Hg derivative.. M. 500 ng bacterial His6-Cdc34. and were frozen. A.. wt&8 #-catenin peptides were conjugated to Rev.. T. wt%4. 2003 Coordinates have been deposited in the RCSB Protein Data Bank Revised: April 14.. Dissociation Constants of !-TrCP1-Skp1-!-Catenin Complexes Haas. K. M. and 85 to 160 of Skp1. Kispert. P.5 hr before being by Ni-NTA affinity and cation exchange chromatography. 2003 In Vitro Ubiquitination Assay of Full-Length I"B# by the In Vitro Translated !-TrCP1 Mutants References I$B" ubiquitination assays were performed at 30(C (1 hr) in a total volume of 15 +l containing 50 ng His6-E1 purified from budding Aberle. The CCP4 chromatography.E. del los Santos. 9. and the NIH.. L. complex and to assemble with the Skp1-Skp2-Cks1 to ubiquitinate Desterro.. Cell 87. Wilchek.. and Sigler. Critical ern blotting using polyclonal antibodies against I$B" (Santa Cruz). wt&4. 44 to 65. was previously shown to have Deshaies.. .. P. The human Cul1-Rbx1 complex 760–763. The free and bound peptides were separated The structure of the complex was determined by a combination of by native-gel electrophoresis using 4. and residues 19 to 29 and 41 to 44 of #-catenin are not visible in the electron density maps and are presumed to be disordered. EMBO 1 +M ubiquitin-aldehyde. Nilges.. 1994) was used binding equilibrium with the program GraFit to obtain KD values and to locate the Skp1 and WD40 portions of the complex with search standard deviations (Figure 5F). The split Cul1.. 376–379.T. J. H. I. cation exchange..and Creighton. R. Albert. type. (1996). J. Perret. residues 2 to 37. and Hay. Ub R7 (200 A complex of Cdc4p.B. Reflection data were indexed. Biochem. A.L. used in the assays consists of the noncovalently associated N. (1998). the F-box. J. Clore. Katchalski-Katzir.. Distribution of end-to-end distances of oligopeptide in solution as dependent-protein kinase (PKA) site followed by a His6 tag (referred estimated by energy transfer. 67. and Stenberg. 577–588. respectively. which allows the production of the Cul1-Rbx1 complex in a soluble form in E. Correll. 207–210. 20 +M LLNL Bai. residues 66 to 70. A. Goebl. and the substrate peptides (170 +M) were mixed in a solution nation of the phosphorylated CDK inhibitor Sic1p. M. and Cdc53p/cullin catalyzes ubiquiti- +M). and the Arthur of #-TrCP1. Cell 91. J...S. 1996). buffer. Chem. DeLano. The R7 mutant of ubiquitin was expressed with an N-terminal His6 Grosse-Kunstieve. (1997). R. Acta Crys- glutathione affinity chromatography. and was visualized by Coomassie staining.C. 1999a). and the average Durand. (NTD&CTD)-Rbx1 (1.. 2002). All proteins were purified to !90% 233–239. Cell 25 mM Tris-HCl (pH 8. coli. I.. #-catenin is a target for the ubiquitin-proteasome pathway.8% polyacrylamide gels in molecular replacement and multiwavelength anomalous diffraction TBE buffer. A. Hershko. and Siebenlist. Rev.H. Crystallography and NMR system: a new soft- pressed in insect cells as a GST-fusion protein and was purified by ware suite for macromolecular structure determination. Ni-NTA affinity and cation exchange chromotography. and (Sigma). C. R.. and tag and purified by Ni-NTA chromatography.P. 435–467. Human E1 was ex. Baldi. 263–274. K. Skp1 connects cell cycle regulators to the ubi- #-TrCP1 proteins or control reticulocyte extracts lacking #-TrCP1. 2002). role for lysines 21 and 22 in signal-induced.. and Rochelle Belfer Foundation. 3797–3804. followed by thrombin cleavage. 905–921. #-TrCP1-Skp1 (1.. J... W... 2003 Published: June 19. Jiang. and 161 to 163 of Skp1. and scaled using 32 P labeled using 32P-ATP and PKA (Sigma). Gros. Skp1p. S. 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