The SCF Ubiquitin Ligase

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The SCF Ubiquitin Ligase: An Extended Look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCFSkp2 ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished. The deluge of recent findings on ubiquitin-dependent proteolysis in diverse areas of cell regulation has stimulated some surprising scientific interactions. At a recent meeting, a furious discourse erupted where a cellular immunologist, an invertebrate developmental geneticist, and a neurobiologist were debating a ubiquitin enzymologist about the previously arcane topic of the dynamics of ubiquitin chain assembly. Unthinkable! As we are finding that the destruction of proteins is restricted in some cases to specific cellular sites and at very specific times in cellular processes—often critically linked to the inactivation or activation of those processes—the mechanics of how this exquisite selectivity is achieved becomes a central question. For some time, the basic model of substrate protein ubiquitination by a shuttle of E1, E2, and E3 enzymes has been fairly clear (Hershko and Ciechanover, 1998). However, the molecular identification of the E3 ubiquitin ligases as proteins or protein complexes containing protein-protein interaction domains for substrate binding and separate domains that couple to the ubiquitin-charged E2 (ubiquitin conjugating) enzyme provided the conceptual bridge between substrate recognition and the catalytic steps for ubiquitin chain formation (for review, see Jackson et al., 2000). In the largest class of E3 ubiquitin ligases, a zinc binding RING finger domain interacts with the E2 enzyme to stimulate ubiquitin chain formation. These RING finger E3 enzymes are formed by either a single polypeptide containing both RING finger and substrate binding domains or by a multicomponent complex with separate polypeptides containing these domains. The best characterized of these multicomponent complexes are the SCF E3 ubiquitin ligases, a highly diverse family of complexes named for its constituents, the Skp1, Cullin, and F box proteins (Deshaies, 1999). Published in the April 18 issue of Nature, the recent crystal structure from the Pavletich laboratory of the SCFSkp2 complex provides a stunning view of its protein topology and offers some clues as to how ubiquitination itself might occur (Zheng et al., 2002). The functional topology of SCF complexes was established by structure-function studies on the prototypes for the SCF complex: the human SCFSkp2 and yeast SCFCdc4 complexes, as well as other related complexes (Jackson et al., 2000; Deshaies, 1999). In the case of the Skp2 complex (represented in a model in the lower

panel of the Figure), an E2 enzyme (yellow) couples to the small Roc1/Rbx1 RING finger protein (red), which in turn associates with the C terminus of the cullin, Cul1 (green). The Cul1 N terminus recruits the highly conserved Skp1 protein (blue), which then binds to the central F box motif in the F box protein Skp2 (pink). Skp2 contains a C-terminal leucine-rich repeat (LRR) domain, which is important for binding its substrate, the Cdk2 inhibitor p27Kip1. p27 must be phosphorylated by cyclin E/Cdk2 before binding to Skp2, and the Cdk2 binding protein Cks1 is involved in the binding reaction (Harper, 2001). An earlier structure from the Pavletich lab contained Skp1 and a large portion of the Skp2 protein including the LRR (Schulman et al., 2000). LRR domains have been broadly implicated in protein-protein interactions, but how and to what site the phosphorylated p27 binds Skp2 is not clear. The Cdc4 protein from the related yeast SCF also binds a phosphorylated Cdk inhibitor, but uses instead a C-terminal WD40, ␤-propeller domain. There are more than a dozen F box proteins containing LRR and WD40 domains, and some are known to bind several substrates, but the presentation and binding of substrates remains one of the most puzzling questions in E3 biochemistry. The newest SCF structure (see Figure, upper panel) from Pavletich’s group (Zheng et al., 2002) contains the full-length versions of Rbx1/Roc1 (red), Cul1 (green), and Skp1 (blue) and a fragment of the Skp2 protein (pink). The complex is organized by the cullin, which interacts with all three subunits and serves as a scaffold. The cullin itself has an N-terminal extended, stalk-like ␣-helical domain of 415 amino acids—comprised of three repeats (cullin repeats) of a novel five-helix bundle—and a 360 amino acid C-terminal domain. The three 120 amino acid cullin repeats are not discernable as repeats from the primary sequence but nevertheless form an extended structure in which each repeat packs against the previous repeat with an amazingly regular 37 A˚ translation and 27⬚ rotation, creating a stalk of over 100 A˚. This long stalk defines the long axis of the SCF complex, but the significance of this extended conformation is still a puzzle. The first repeat is altered slightly to form a binding site for the Skp1 protein. This site is conserved among Cul1 orthologs, but not among the other cullin paralogs (Cul2, 3, 4A, 4B, 5). However, the other cullins show homology with their orthologs within this region, suggesting that this may be the binding site for Skp1 equivalents for these cullins. Notably, sites within the N terminus of Cul2 show conservation that might contribute to binding of the Skp1 homolog and Cul2 binding partner elongin C. The Cul1 CTD is composed of a four-helix bundle (4HB), an ␣/␤ domain, and two copies of a “wingedhelix” motif (WH-A and WH-B). The ␣/␤ domain contains four of the strands of a five-strand ␤ sheet, while the other strand is contributed by the RING finger protein Rbx1. The ␤ strand contributed by Rbx1 is buried deeply in the ␣/␤ domain, suggesting an explanation for the tight association between Rbx1 and Cul1. In the WH-B domain, near the interface where the Rbx1 protein might bind the E2, is the site for modification by the ubiquitinlike molecule Nedd8, although the Nedd8 modification is not present here. This neddylation site is important for SCF activity and may affect the interaction with the

Molecular Cell 924

The Quaternary Structure of the Cul1-Rbx1Skp1-F-boxSkp2 Complex The upper panel shows the structure derived from a partial SCF complex (Zheng et al., 2002). The lower panel shows a model extending this structure to include the majority of the Skp2 protein (from a Skp1-Skp2 structure [Schulman et al., 2000]) and an E2 enzyme (from the structure of a related RING finger protein complexed to an E2 enzyme [Zheng et al., 2000]).

E2 (Deshaies, 1999). The CTD domain is highly conserved among cullins and forms a V-shaped groove to bind the RING finger. The WH-A domain and part of the 4HB and ␣/␤ domains comprise the previously defined cullin homology region (Zachariae et al., 1998; Yu et al., 1998). Though proteins containing a cullin homology domain generally lack primary sequence homology outside of this domain, the highly ordered packing of the final WH-B domain C-terminal to the cullin homology domain suggests this region might be structurally conserved in other cullins. Indeed, the authors crystallize and solve the structure of the analogous C-terminal fragment of yeast APC2, the somewhat distantly related cullin from the Anaphase Promoting Complex (APC), and find this also forms a WH domain! The structure also shows that the 70 residue RING finger from Rbx1 is a variant RING, containing not only the two zinc binding sites typically seen in RING fingers (yellow balls) but also an inserted loop that forms a third zinc binding site. Although of uncertain importance, this site is apparently shared in the APC RING finger protein, APC11 (Tang et al., 2001). The Rbx1 RING also contains a hydrophobic groove similar to that seen in the structure of the Cbl E3 ubiquitin ligase, another RING finger E3 (Zheng et al., 2000). The Cbl structure includes the E2 ubiquitin-conjugating enzyme, which associates di-

rectly with the Cbl RING finger, providing a possible model for other RING finger-E2 interactions. What does the structure demonstrate? A snapshot of the SCF core provides us with close-up views of the interfaces of Cul1 with the other SCF components. This alone provides rich possibilities for additional structurefunction studies. Analysis of the interface between the cullin and the RING finger and E2 proteins may help in understanding how the ubiquitin-charged E2 is recruited and the very mysterious question of how ubiquitin chains are formed. The ability to visualize the Cul1-NTD interaction with Skp1 provides clues about possible adaptor sites for Skp1 homologs in other cullins. A surprising aspect of the structure is the lack of any flexible linkages and the apparent rigidity of the structure. The authors try a simple test of the importance of this rigid scaffold by reengineering the interface between the Cul1-NTD and -CTD with a flexible linker and find that this variant is inactive, although more detailed studies would be needed to truly validate this idea. The authors also use their prior structural knowledge of the Skp1Skp2 interface (Schulman et al., 2000) and the RING finger domain-E2 interface (Zheng et al., 2000) to include the full-length Skp2 and E2 enzymes in a model of the complete SCF complex (see Figure, lower panel). In this modeled complex, the E2 sits atop the Rbx1 protein,

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and the Skp2 LRR points back toward the E2. Though this narrows the gap between the LRR and the E2 to ⵑ50 A˚, this is still a long distance for the E2 to transfer its charged ubiquitin to a substrate bound to Skp2. And while it is possible that substrates are recognized and ubiquitinated over this distance or that the substrate protein is unfolded to be presented to the E2, it is also possible that additional factors must be recruited or higher order complexes must be formed to allow the addition of the first ubiquitin to a side chain lysine of the substrate and the subsequent assembly of a chain. What is also not clear is how a ubiquitin-charged E2 enzyme or an SCF substrate would bind, and thus future structures including the charged E2 or substrate might help us better understand catalysis. So whereas the cullin provides an extended structure to organize the substrate binding and catalytic domains of the SCF complex, an extended look will still be needed to understand how substrates are recognized and ubiquitin chains are formed. Peter K. Jackson and Adam G. Eldridge Stanford University School of Medicine Department of Pathology

RNAi Targeting an Animal Virus: News from the Front Although many eukaryotic organisms exhibit RNA interference, its role as an antiviral pathway is established only for plants. A recent paper demonstrates that it also acts in animals, and reveals a viral suppressor of this process. The emerging field of RNA interference (RNAi) is developing at an exhilarating pace, tying together biologists interested in gene regulation, development, virus-host interactions, plant pathology, and RNA metabolism, via a common, double-stranded thread. RNAi is the targeted destruction of mRNAs via complementarity to double-stranded RNA (dsRNA) activators (see Hutva´gner and Zamore, 2002 for review). It is a key mechanism of posttranscriptional gene silencing (PTGS), which was originally observed in plants. RNAi was identified as the mediator of PTGS in the nematode Caenorhabditis elegans (Fire, 1998), and similar processes have been described for plants, other invertebrate animal systems such as Drosophila melanogaster, some fungi (Neurospora crassa), and mammals, including humans. The use of RNAi as an antiviral defense is well established in plants, which can recover from virus infection and are protected from challenge with homologous viruses. Furthermore, several positive-strand RNA plant viruses have evolved suppressors of the RNAi pathway (see Figure; for reviews, see Li and Ding, 2001; Vance and Vaucheret, 2001), and there is even evidence that

300 Pasteur Drive Palo Alto, California 94305 Selected Reading Deshaies, R.J. (1999). Annu. Rev. Cell. Dev. Biol. 15, 435–467. Harper, J.W. (2001). Curr. Biol. 11, R431–R435. Hershko, A., and Ciechanover, A. (1998). Annu. Rev. Biochem. 67, 425–479. Jackson, P.K., Eldridge, A.G., Freed, E., Furstenthal, L., Hsu, J.Y., Kaiser, B.K., and Reimann, J.D. (2000). Trends Cell Biol. 10, 429–439. 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. Tang, Z., Li, B., Bharadwaj, R., Zhu, H., Ozkan, E., Hakala, K., Deisenhofer, J., and Yu, H. (2001). Mol. Biol. Cell 12, 3839–3851. Yu, H., Peters, J.M., King, R.W., Page, A.M., Hieter, P., and Kirschner, M.W. (1998). Science 279, 1219–1222. Zachariae, W., Shevchenko, A., Andrews, P.D., Ciosk, R., Galova, M., Stark, M.J., Mann, M., and Nasmyth, K. (1998). Science 279, 1216–1219. Zheng, N., Schulman, B.A., Song, L., Miller, J.J., Jeffrey, P.D., Wang, P., Chu, C., Koepp, D.M., Elledge, S.J., Pagano, M., et al. (2002). Nature 416, 703–709. Zheng, N., Wang, P., Jeffrey, P.D., and Pavletich, N.P. (2000). Cell 102, 533–539.

plants have developed ways for subverting these suppressors. In the May 17, 2002 issue of Science, Li et al. describe a viral suppressor of RNAi, the B2 protein of Flock House virus (FHV), an insect pathogen. Their data demonstrate that RNAi also functions as an antiviral response in animals for which the virus has evolved a counter-defense strategy. Intriguingly, this protein suppresses RNAi in plants, further underscoring the deep evolutionary relatedness of this process in disparate organisms. Although a coherent picture for the mechanism of RNAi remains incomplete, numerous genes involved in this process have been identified in plants, nematodes, fruit flies, and fungi and strongly suggest an evolutionarily conserved mechanism (Hutva´gner and Zamore, 2002). Recent progress has also come from biochemical characterization of the RNAi machinery, including the induction of RNAi in extracts of D. melanogaster (Zamore et al., 2000). RNAi is initiated by the ATP-dependent cleavage of dsRNA into 21–23 nt duplexes, termed small interfering RNA (siRNA) by the RNase III-like enzyme Dicer. At least in Drosophila, individual strands of siRNA are incorporated into a ribonucleoprotein RNAinduced silencing complex (RISC) and target complementary RNAs for degradation by an uncharacterized RISC-associated RNase. Several lines of evidence also point to a role for an RNA-dependent RNA polymerase in RNAi induction or maintenance for some organisms. Ding’s group had previously established that the 2b protein of cucumber mosaic virus, a positive strand RNA virus of plants, functions as a suppressor of PTGS (Li and Ding, 2001). Positive-strand RNA viruses are an important class of plant and animal pathogens, including the agents responsible for such human diseases as po-