Structural Analysis of Siah1-Siah-interacting Protein Interactions and Insights into the Assembly of an E3 Ligase Multiprotein Complex

Abstract

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets β-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating β-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex. Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets β-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating β-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex. Polyubiquitination of specific proteins in cells involves the concerted action of E1, 5The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; SCF, Skp1-cullin-1-F-box; SIP, Siah-interacting protein; CAPS, 3-(cyclohexylamino)propanesulfonic acid; TROSY, transverse relaxation optimized spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy. E2, and E3 enzymes. First, E1 covalently binds and activates ubiquitin for subsequent transfer to one of several E2s. The latter can in turn directly transfer its bound ubiquitin to the amino groups of lysine side chains in target proteins. More often, however, E3 ligases recognize substrates and direct their interaction with E2s, resulting in the highly specific regulation of target protein polyubiquitination (1Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1200) Google Scholar, 2Varshavsky A. Trends Biochem. 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Siah1 expression is up-regulated by p53, revealing a link between genotoxic injury and destruction of β-catenin, reduced Tcf/LEF activity, and cell cycle arrest (31Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Siah1 is a dimeric protein that contains an N-terminal RING domain (an E2 binding domain) followed by two zinc finger motifs and a C-terminal dimerization domain. The crystal structure of a major fragment of Siah1a lacking the RING domain (Siah1ΔR) has been determined (32Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (117) Google Scholar); in this structure, the first zinc finger is highly mobile, whereas the against the C-terminal a dimeric domain. In the multiprotein E3 complex, Siah1 an analogous to the and in the SCF complex, and Siah-interacting protein provides a novel link between Siah1 and Skp1 (31Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). that SIP binds to Siah1 in its N-terminal whereas its binds to Skp1 (31Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). The of SIP is as a protein A. U. Biochem. J. PubMed Scopus Google Scholar, 34Filipek A. Kuznicki J. J. Neurochem. 1998; 70: 1793-1798Crossref PubMed Scopus (94) Google Scholar). binding does not appear to with Skp1 and its functional has not been consensus motif for Siah-binding the has been C.M. Frew I.J. G. Traficante N. E. Bowtell D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). motif is in of Siah-binding of which are targets of degradation. 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Google Scholar). and structure of been in the with of with and and used for a and and for and been in the a with a and and transverse relaxation optimized with or and a of or and to and a of in in the a of in the of of as with and to and with of and both and in and and by the with and with C. T. M. K. J. PubMed Scopus Google and the as a of Siah1 and to the is the of the is the in the of Siah1, and is the as of the with the to the C. T. M. K. J. PubMed Scopus Google Scholar). to the the of and is the from the G.M. R. J. Chem. 1998; 19: Scopus Google used for the used the for The of Siah1 and SIP to domain as a The as of of and of of of of of of and a of and Cell in SIP by a SIP (31Matsuzawa S. Reed J.C. Mol. 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Biol. 2000; 7: PubMed Scopus Google Scholar). of The one the of a of the with in the C-terminal of with In addition, several within the domain are to a of interaction contains the of the consensus Siah-binding within its we a of in which its are and the are and in the whereas all are with and of a in the to groups of and groups of two amino in both the domain and the the in their with for the in the that are to the whereas the side chains of a for the in the motif in binding to we to with either SIP or we in in complex with a SIP The binds to with a of as determined by the structure by The crystal and packing are for the the structure and are and as in the structure, the first zinc finger is is for the and is for one of the motif NOE a that the binds to and an extended as by that from to SIP binds of the saddle-shaped upper surface of the Siah1 which is formed by an with the of the two by motif a that the from to with of in addition, the central a to in part of the lower of the Siah The side chains of the consensus and against the the of the The of the an from of this is the between the and the zinc finger with the structure, binding in the structure of in this The against the side of in and into with their of Siah-binding proteins. from and been to to Siah1 and to with a of Siah-binding SIP C.M. Frew I.J. G. Traficante N. E. Bowtell D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). The and the central motif that been to for that two Siah1 and are to with the their The PXAXVXP for with has been that of the within the and reduced the binding of SIP to Siah1 in an in C.M. Frew I.J. G. Traficante N. E. Bowtell D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). the of by the binding of SIP and Siah1 in in cells. in Siah1 with not with of Siah degradation and of β-catenin in which can by of of SIP (31Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). of this by β-catenin in cells with Siah1 and or cells with Siah1, or in with or in of Siah1 a in of of SIP with Siah1 β-catenin SIP is in cells. In a SIP with in the Siah-binding as a the of Siah1, β-catenin is a for the Tcf/LEF M. P. 2000; PubMed Scopus Google Scholar), the of and SIP Tcf/LEF V. N. D. R. H. 1997; 275: PubMed Scopus Google Scholar, V. N. H. 1997; 275: PubMed Scopus Google Scholar). with β-catenin a in Tcf/LEF in and of an of a reduced by of SIP with Siah1 not to a in Tcf/LEF In of SIP to of Tcf/LEF and of the show that and are required for interaction of SIP with Siah1 in and for the of the proteins in cells. The SIP across the Siah1 of the with their the two of the central of the of the SIP N-terminal dimerization domain. of to that the SIP dimerization domain its to across the formed by the the upper surface of show that the SIP by binding to Siah1 to its lower surface and we by that the surface of the Siah1 are for S. Li C. Takayama S. Reed J.C. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). this the of the SIP to a the surface of the In the the surface of the Siah1 with the of SIP and Siah1 this we and from the surface of the dimerization domain of single a a reduced Siah1 binding However, a binding to in The is as by its to and to with SIP a for the SIP N-terminal domain in the interaction with Siah1 in addition to its as a dimerization The for the of the interaction for β-catenin regulation in we the of the SIP, β-catenin in in cells. of Siah1 reduced of β-catenin of SIP with Siah1 not degradation of β-catenin, SIP is in cells. In SIP the of Siah1 acting in a in a of SIP with Siah1 the of Tcf/LEF with Siah1 whereas of SIP with Siah1 the of Siah1, Tcf/LEF that a is required for the and of the complex. of Siah1 the of the E3 of the the lower surface of Siah1 from the that binds the SIP dimerization domain) to the zinc finger domain and several that the C-terminal domain of SIP, contains the binding for Skp1 (31Matsuzawa S. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). the that this lower surface is involved in of the E3 multiprotein complex, we in two of the surface The the to SIP, as by and and that not However, the to degradation of β-catenin or to and that a functional in the to the SCF multiprotein E3 ligase complex, the complex is to by a scaffold that the by bringing the E2 enzyme and its substrate into apposition with the to a of the structure and of the multiprotein Siah1 complex. two binding for SIP Siah1, both of which are for the complex to and that of either of two functional in a that the interaction is required for the of the E3 ligase complex. The N-terminal domain of a and are with a in which the sits the formed by the the upper surface of Siah1, two the against both sides of the Siah1 specific The binding in which a from the a is of the between and and their as as of the related tumor factor factor with (32Polekhina G. House C.M. Traficante N. Mackay J.P. Relaix F. Sassoon D.A. Parker M.W. Bowtell D.D. Nat. Struct. Biol. 2002; 9: 68-75Crossref PubMed Scopus (117) Google Scholar, C. E. He J. D. Reed J.C. G. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The between the of the SIP and the of the SIP can by the a for the of the PXAXVXP motif in More of Siah-binding proteins this motif from and the motif been to with a of Siah-binding SIP C.M. Frew I.J. G. Traficante N. E. Bowtell D.D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). that Siah-binding proteins in the that we for The of this interaction is by of the AF4 which is in leukemia J. L. Proc. Natl. Acad. Sci. U. S. A. 90: PubMed Scopus Google and in the P.L. Jones E.L. A. A. L. P. Davies K.E. J. 2003; 23: PubMed Google Scholar). The AF4 binds Siah1 A. Moritz S. Gaussmann A. Dingermann T. Marschalek R. 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In of the of the surface of the Siah1 the side from the binding for the SIP dimerization domain. that the C-terminal domain of SIP and its Skp1 to this lower surface that are highly in the Siah1 crystal by crystal as as an that against the that of one of the as as of a the surface of β-catenin degradation SIP Thus, a for the lower surface of Siah1 in the E3 ligase into a functional complex. a in a with of Siah1 and to the is to the packing of against the Cho Oncogene. 2004; 23: PubMed Scopus Google Scholar). we that the binding of SIP for Siah1 is with the of from to this we propose that the between of a single protein E3 ligase an multiprotein complex the of between SIP and substrate proteins binding the Siah1 and by the of of the multiprotein complex that and for in and for the of and of and their for to the