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• W2895550411 abstract "The NEDD4-2 (neural precursor cell–expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin–binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT–ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2. The NEDD4-2 (neural precursor cell–expressed developmentally down-regulated 4-2) HECT ligase catalyzes polyubiquitin chain assembly by an ordered two-step mechanism requiring two functionally distinct E2∼ubiquitin–binding sites, analogous to the trimeric E6AP/UBE3A HECT ligase. This conserved catalytic mechanism suggests that NEDD4-2, and presumably all HECT ligases, requires oligomerization to catalyze polyubiquitin chain assembly. To explore this hypothesis, we examined the catalytic mechanism of NEDD4-2 through the use of biochemically defined kinetic assays examining rates of 125I-labeled polyubiquitin chain assembly and biophysical techniques. The results from gel filtration chromatography and dynamic light-scattering analyses demonstrate for the first time that active NEDD4-2 is a trimer. Homology modeling to E6AP revealed that the predicted intersubunit interface has an absolutely conserved Phe-823, substitution of which destabilized the trimer and resulted in a ≥104-fold decrease in kcat for polyubiquitin chain assembly. The small-molecule Phe-823 mimic, N-acetylphenylalanyl-amide, acted as a noncompetitive inhibitor (Ki = 8 ± 1.2 mm) of polyubiquitin chain elongation by destabilizing the active trimer, suggesting a mechanism for therapeutically targeting HECT ligases. Additional kinetic experiments indicated that monomeric NEDD4-2 catalyzes only HECT∼ubiquitin thioester formation and monoubiquitination, whereas polyubiquitin chain assembly requires NEDD4-2 oligomerization. These results provide evidence that the previously identified sites 1 and 2 of NEDD4-2 function in trans to support chain elongation, explicating the requirement for oligomerization. Finally, we identified a conserved catalytic ensemble comprising Glu-646 and Arg-604 that supports HECT–ubiquitin thioester exchange and isopeptide bond formation at the active-site Cys-922 of NEDD4-2. The HECT 2The abbreviations used are: HECThomologous to E6AP C terminusE6APE6-associated proteinENaCepithelial sodium channelGSTglutathione S-transferaseIsoTisopeptidase T. family of ubiquitin ligases consists of 28 function-specific paralogs in humans (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 2Metzger M.B. Hristova V.A. Weissman A.M. HECT and RING finger families of E3 ubiquitin ligases at a glance.J. Cell Sci. 2012; 125 (22389392): 531-53710.1242/jcs.091777Crossref PubMed Scopus (426) Google Scholar3Lorenz S. Structural mechanisms of HECT-type ubiquitin ligases.Biol. Chem. 2018; 399 (29016349): 127-14510.1515/hsz-2017-0184Crossref PubMed Scopus (70) Google Scholar). Compared with the ∼600-member superfamily of RING (really interesting new gene) ligases, the relatively small number of HECT ligases plays integral but disproportionate roles in diverse cellular signaling pathways, disruption of which results in a spectrum of pathological conditions (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 4Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78 (19489725): 399-43410.1146/annurev.biochem.78.101807.093809Crossref PubMed Scopus (1875) Google Scholar, 5Grau-Bové X. Sebe-Pedros A. Ruiz-Trillo I. A genomic survey of HECT ubiquitin ligases in eukaryotes reveals independent expansions of the HECT system in several lineages.Genome Biol. Evol. 2013; 5 (23563970): 833-84710.1093/gbe/evt052Crossref PubMed Scopus (26) Google Scholar). Members of the HECT family are approximately 100 kDa in molecular mass and are defined by the presence of a highly conserved 350-residue C-terminal domain responsible for binding its cognate E2∼ubiquitin 3The tilde symbol (∼) denotes a high-energy thioester bond. thioester and catalyzing the formation of a high-energy HECT∼ubiquitin thioester intermediate prior to conjugation of the activated ubiquitin to the target protein (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 3Lorenz S. Structural mechanisms of HECT-type ubiquitin ligases.Biol. Chem. 2018; 399 (29016349): 127-14510.1515/hsz-2017-0184Crossref PubMed Scopus (70) Google Scholar). Formation of the HECT∼ubiquitin covalent intermediate distinguishes this family of conjugating enzymes from the RING ligases that attach ubiquitin to protein targets directly from the E3-bound E2∼ubiquitin thioester co-substrate (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 2Metzger M.B. Hristova V.A. Weissman A.M. HECT and RING finger families of E3 ubiquitin ligases at a glance.J. Cell Sci. 2012; 125 (22389392): 531-53710.1242/jcs.091777Crossref PubMed Scopus (426) Google Scholar, 4Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78 (19489725): 399-43410.1146/annurev.biochem.78.101807.093809Crossref PubMed Scopus (1875) Google Scholar). homologous to E6AP C terminus E6-associated protein epithelial sodium channel glutathione S-transferase isopeptidase T. The HECT superfamily can be subdivided according to differences in the N-terminal domain architecture (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 3Lorenz S. Structural mechanisms of HECT-type ubiquitin ligases.Biol. Chem. 2018; 399 (29016349): 127-14510.1515/hsz-2017-0184Crossref PubMed Scopus (70) Google Scholar). The NEDD4 family of HECT ligases consists of nine members characterized by an N-terminal C2 domain responsible for membrane anchoring and 2–4 WW domains that bind PY motifs present on target proteins to recruit the latter to the catalytic domain (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar). The NEDD4 ligases are key mediators in protein trafficking of transmembrane receptors and ion channels (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 6Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.EMBO J. 1996; 15 (8665844): 2371-238010.1002/j.1460-2075.1996.tb00593.xCrossref PubMed Scopus (739) Google Scholar7Zhou R. Patel S.V. Snyder P.M. Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.J. Biol. Chem. 2007; 282 (17502380): 20207-2021210.1074/jbc.M611329200Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 8Rotin D. Staub O. Haguenauer-Tsapis R. Ubiquitination and endocytosis of plasma membrane proteins: Role of Nedd4/Rsp5p family of ubiquitin-protein ligases.J. Membr. Biol. 2000; 176 (10882424): 1-1710.1007/s0023200107910.1007/s002320001079Crossref PubMed Google Scholar9Hicke L. Dunn R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.Annu. Rev. Cell Dev. Biol. 2003; 19 (14570567): 141-17210.1146/annurev.cellbio.19.110701.154617Crossref PubMed Scopus (960) Google Scholar). Their role in facilitating vesicular transport has been exploited by the Ebola and Marburg viruses to promote viral egress (1Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10 (19436320): 398-40910.1038/nrm2690Crossref PubMed Scopus (766) Google Scholar, 10Morita E. Sundquist W.I. Retrovirus budding.Annu. Rev. Cell Dev. Biol. 2004; 20 (15473846): 395-42510.1146/annurev.cellbio.20.010403.102350Crossref PubMed Scopus (548) Google Scholar, 11Freed E.O. Viral late domains.J. Virol. 2002; 76 (11967285): 4679-468710.1128/JVI.76.10.4679-4687.2002Crossref PubMed Scopus (378) Google Scholar12Han Z. Sagum C.A. Bedford M.T. Sidhu S.S. Sudol M. Harty R.N. ITCH E3 ubiquitin ligase interacts with Ebola virus VP40 to regulate budding.J. Virol. 2016; 90 (27489272): 9163-917110.1128/JVI.01078-16Crossref PubMed Scopus (43) Google Scholar). The NEDD4-2 (neural precursor cell–expressed developmentally down-regulated 4-2) paralog is the best characterized member of the family and is noted for catalyzing Lys-63-linked polyubiquitination of the amelioride-sensitive epithelial sodium channel (ENaC) in the distal nephrons to promote endocytic uptake and lysosomal degradation (6Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.EMBO J. 1996; 15 (8665844): 2371-238010.1002/j.1460-2075.1996.tb00593.xCrossref PubMed Scopus (739) Google Scholar, 7Zhou R. Patel S.V. Snyder P.M. Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.J. Biol. Chem. 2007; 282 (17502380): 20207-2021210.1074/jbc.M611329200Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 13Goel P. Manning J.A. Kumar S. NEDD4-2 (NEDD4L): The ubiquitin ligase for multiple membrane proteins.Gene. 2015; 557 (25433090): 1-1010.1016/j.gene.2014.11.051Crossref PubMed Scopus (99) Google Scholar, 14Kabra R. Knight K.K. Zhou R. Snyder P.M. Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.J. Biol. Chem. 2008; 283 (18174164): 6033-603910.1074/jbc.M708555200Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Disruption of NEDD4-2–dependent ENaC targeting results in Liddle’s syndrome, a rare autosomal dominant salt-sensitive hypertension (6Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.EMBO J. 1996; 15 (8665844): 2371-238010.1002/j.1460-2075.1996.tb00593.xCrossref PubMed Scopus (739) Google Scholar, 7Zhou R. Patel S.V. Snyder P.M. Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.J. Biol. Chem. 2007; 282 (17502380): 20207-2021210.1074/jbc.M611329200Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 15Lifton R.P. Gharavi A.G. Geller D.S. Molecular mechanisms of human hypertension.Cell. 2001; 104 (11239411): 545-55610.1016/S0092-8674(01)00241-0Abstract Full Text Full Text PDF PubMed Scopus (1365) Google Scholar, 16Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome.J. Clin. Invest. 1999; 103 (10074483): 667-67310.1172/JCI5713Crossref PubMed Scopus (326) Google Scholar17Lu C. Pribanic S. Debonneville A. Jiang C. Rotin D. The PY motif of ENaC, mutated in Liddle syndrome, regulates channel internalization, sorting and mobilization from subapical pool.Traffic. 2007; 8 (17605762): 1246-126410.1111/j.1600-0854.2007.00602.xCrossref PubMed Scopus (99) Google Scholar). The NEDD4-2 ligase also targets voltage-gated sodium channels (Nasv) in vitro and in vivo, disruption of which is linked to Nasv-dependent hyperexcitability and neuropathic pain, along with the development of mesial temporal lobe epilepsy (18Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2.J. Biol. Chem. 2004; 279 (15123669): 28930-2893510.1074/jbc.M402820200Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar19Laedermann C.J. Cachemaille M. Kirschmann G. Pertin M. Gosselin R.D. Chang I. Albesa M. Towne C. Schneider B.L. Kellenberger S. Abriel H. Decosterd I. Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.J. Clin. Invest. 2013; 123 (23778145): 3002-301310.1172/JCI68996Crossref PubMed Scopus (85) Google Scholar, 20Laedermann C.J. Decosterd I. Abriel H. Ubiquitylation of voltage-gated sodium channels.Handb. Exp. Pharmacol. 2014; 221 (24737239): 231-25010.1007/978-3-642-41588-3_11Crossref PubMed Scopus (19) Google Scholar21Wu L. Peng J. Kong H. Yang P. He F. Deng X. Gan N. Yin F. The role of ubiquitin/Nedd4-2 in the pathogenesis of mesial temporal lobe epilepsy.Physiol. Behav. 2015; 143 (25700894): 104-11210.1016/j.physbeh.2015.02.026Crossref PubMed Scopus (14) Google Scholar). In contrast, knockdown of NEDD4-2 in murine lung epithelial cells results in a cystic fibrosis–like disease, consistent with its role in regulating ENaC and ΔF508-CFTR protein levels at the membrane (22Kimura T. Kawabe H. Jiang C. Zhang W. Xiang Y.Y. Lu C. Salter M.W. Brose N. Lu W.Y. Rotin D. Deletion of the ubiquitin ligase Nedd4L in lung epithelia causes cystic fibrosis-like disease.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21300902): 3216-322110.1073/pnas.1010334108Crossref PubMed Scopus (82) Google Scholar, 23Caohuy H. Jozwik C. Pollard H.B. Rescue of ΔF508-CFTR by the SGK1/Nedd4-2 signaling pathway.J. Biol. Chem. 2009; 284 (19617352): 25241-2525310.1074/jbc.M109.035345Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The morbidity and mortality associated with the disruption of NEDD4-2–dependent cell signaling highlight the importance of the NEDD4 family in cell homeostasis. Unfortunately, our knowledge of the roles the HECT ligases assume in cellular regulation exceeds our understanding of the mechanism(s) by which these enzymes function. Early structural and biochemical studies supported a standard model for HECT ligase polyubiquitin chain assembly through a distal sequential addition mechanism as a reiterative extension of substrate monoubiquitination (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar, 25Kamadurai H.B. Souphron J. Scott D.C. Duda D.M. Miller D.J. Stringer D. Piper R.C. Schulman B.A. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex.Mol. Cell. 2009; 36 (20064473): 1095-110210.1016/j.molcel.2009.11.010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar26Hochstrasser M. Lingering mysteries of ubiquitin-chain assembly.Cell. 2006; 124 (16413479): 27-3410.1016/j.cell.2005.12.025Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In this model, the cognate E2∼ubiquitin co-substrate binds to a single site at the small N-terminal subdomain of the HECT catalytic module prior to transthiolation to the HECT active-site cysteine, suggested by the crystal structure of the isolated E6AP HECT domain in complex with UbcH7 solved by Huang et al. (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar). The resulting HECT-linked ubiquitin thioester is then transferred to lysine side chain(s) present on the target protein to form an isopeptide bond in a step termed monoubiquitination (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar). Subsequent chain elongation is thought to proceed by the addition of ubiquitin moieties to the distal end of the growing polyubiquitin chain through an iterative sequence of steps (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar, 25Kamadurai H.B. Souphron J. Scott D.C. Duda D.M. Miller D.J. Stringer D. Piper R.C. Schulman B.A. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex.Mol. Cell. 2009; 36 (20064473): 1095-110210.1016/j.molcel.2009.11.010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The E6AP HECT–UbcH7 structure reveals an approximately 41-Å gap between the donor and acceptor sites even though the two sulfur atoms are required to approach within atomic distance to support nucleophilic attack during thioester exchange (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar). Kamadurai et al. (25Kamadurai H.B. Souphron J. Scott D.C. Duda D.M. Miller D.J. Stringer D. Piper R.C. Schulman B.A. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex.Mol. Cell. 2009; 36 (20064473): 1095-110210.1016/j.molcel.2009.11.010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) attempted to resolve this paradox by solving the crystal structure of the NEDD4-2HECT domain in complex with a Ubc5BL3S/T98K double mutant covalently linked to ubiquitin to simulate the otherwise labile thioester bond. The latter structure demonstrated similar E2 binding at the small N-terminal subdomain identified for E6AP, and rotation of the C-terminal subdomain harboring the HECT active-site cysteine reduced the donor-acceptor distance to 8 Å, seemingly resolving the topological barrier to facile transthiolation (25Kamadurai H.B. Souphron J. Scott D.C. Duda D.M. Miller D.J. Stringer D. Piper R.C. Schulman B.A. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex.Mol. Cell. 2009; 36 (20064473): 1095-110210.1016/j.molcel.2009.11.010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Crystal structures of other HECT domains possess similar architectures, with the HECT C-terminal subdomain adopting a variety of orientations relative to the N-terminal subdomain (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar, 25Kamadurai H.B. Souphron J. Scott D.C. Duda D.M. Miller D.J. Stringer D. Piper R.C. Schulman B.A. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex.Mol. Cell. 2009; 36 (20064473): 1095-110210.1016/j.molcel.2009.11.010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 27Ogunjimi A.A. Briant D.J. Pece-Barbara N. 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Biol. Commun. 2015; 71 (26457515): 1251-125710.1107/S2053230X1501554XCrossref PubMed Scopus (13) Google Scholar). These observations have led to the gradual acceptance of a mechanism predicated on large conformational changes of the HECT C-terminal subdomain to close the gap required for thioester exchange (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar, 25Kamadurai H.B. Souphron J. Scott D.C. Duda D.M. Miller D.J. Stringer D. Piper R.C. Schulman B.A. Insights into ubiquitin transfer cascades from a structure of a UbcH5B∼ubiquitin-HECT(NEDD4L) complex.Mol. Cell. 2009; 36 (20064473): 1095-110210.1016/j.molcel.2009.11.010Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 28Verdecia M.A. Joazeiro C.A. Wells N.J. Ferrer J.L. Bowman M.E. Hunter T. Noel J.P. Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase.Mol. Cell. 2003; 11 (12535537): 249-25910.1016/S1097-2765(02)00774-8Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Verdecia et al. (28Verdecia M.A. Joazeiro C.A. Wells N.J. Ferrer J.L. Bowman M.E. Hunter T. Noel J.P. Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase.Mol. Cell. 2003; 11 (12535537): 249-25910.1016/S1097-2765(02)00774-8Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) observed that modifying the flexible linker connecting the N- and C-terminal subdomains by substitution of prolyl residues significantly decreases the activity of the WWP1 HECT ligase, interpreted as restricting the required mobility proposed for the C-terminal subdomain. In contrast, more recent studies by Ronchi et al. (31Ronchi V.P. Klein J.M. Edwards D.J. Haas A.L. The active form of E6-associated protein (E6AP)/UBE3A ubiquitin ligase is an oligomer.J. Biol. Chem. 2014; 289 (24273172): 1033-104810.1074/jbc.M113.517805Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 32Ronchi V.P. Klein J.M. Haas A.L. E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites.J. Biol. Chem. 2013; 288 (23439649): 10349-1036010.1074/jbc.M113.458059Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar33Ronchi V.P. Summa C.M. Kim E.D. Klein J.M. Haas A.L. In silico modeling of the cryptic E2∼ubiquitin binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.J. Biol. Chem. 2017; 292 (28924046): 18006-18023Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) for the first time employing biochemically defined kinetic assays of full-length E6AP-catalyzed polyubiquitin chain formation have revealed marked inconsistencies that directly rule out the standard model. These new observations proffer a two-site proximal indexation mechanism that is distinguished by the coordinated sequential binding of E2∼ubiquitin at functionally distinct E2-binding sites that assemble Lys-48–linked polyubiquitin chains in an “inside-out” manner on the HECT active-site cysteine prior to stochastic transfer of the preassembled degradation signal en bloc to a target substrate or competing nucleophile such as water (31Ronchi V.P. Klein J.M. Edwards D.J. Haas A.L. The active form of E6-associated protein (E6AP)/UBE3A ubiquitin ligase is an oligomer.J. Biol. Chem. 2014; 289 (24273172): 1033-104810.1074/jbc.M113.517805Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 32Ronchi V.P. Klein J.M. Haas A.L. E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites.J. Biol. Chem. 2013; 288 (23439649): 10349-1036010.1074/jbc.M113.458059Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar33Ronchi V.P. Summa C.M. Kim E.D. Klein J.M. Haas A.L. In silico modeling of the cryptic E2∼ubiquitin binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.J. Biol. Chem. 2017; 292 (28924046): 18006-18023Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). According to this model, the initial binding of E2∼ubiquitin at a cryptic site 1, not observed in the original Huang et al. (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar) structure but required by the more recent kinetic data, is associated with thioester exchange to yield the HECT∼ubiquitin intermediate followed by binding of a second E2∼ubiquitin at the canonical E2-binding site (site 2) to support polyubiquitin chain elongation (24Huang L. Kinnucan E. Wang G. Beaudenon S. Howley P.M. Huibregtse J.M. Pavletich N.P. Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade.Science. 1999; 286 (10558980): 1321-132610.1126/science.286.5443.1321Crossref PubMed Scopus (439) Google Scholar, 32Ronchi V.P. Klein J.M. Haas A.L. E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites.J. Biol. Chem. 2013; 288 (23439649): 10349-1036010.1074/jbc.M113.458059Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 33Ronchi V.P. Summa C.M. Kim E.D. Klein J.M. Haas A.L. In silico modeling of the cryptic E2∼ubiquitin binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.J. Biol. Chem. 2017; 292 (28924046): 18006-18023Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). More recently, parallel studies have demonstrated this two-site kinetic mechanism in the paralogous full-length NEDD4-2HECT ligase that assembles Lys-63–linked polyubiquitin chains, suggesting conservation across the superfamily (34Todaro D.R. Augustus-Wallace A.C. Klein J.M. Haas A.L. The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.J. Biol. Chem. 2017; 292 (28972136): 19521-1953610.1074/jbc.M117.817882Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). A concurrent observation that the evolutionarily unrelated IpaH and SspH2 families of bacterial ubiquitin ligases exhibit an analogous mechanism suggests convergent evolution, which provides strong circumstantial support for the conservation of the proximal indexation mechanism (35Edwards D.J. Streich Jr, F.C. Ronchi V.P. Todaro D.R. Haas A.L. Convergent evolution in the assembly of polyubiquitin degradation signals by the Shigella flexneri IpaH9.8 ligase.J. Biol. Chem. 2014; 289 (25342744): 34114-3412810.1074/jbc.M114.609164Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). These observations expand on an original observation by Wang and Pickart (36Wang M. Pickart C.M. Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis.EMBO J. 2005; 24 (16341092): 4324-433310.1038/sj.emboj.7600895Crossref PubMed Scopus (101) Google Scholar) suggesting that the isolated E6AP HECT domain might assemble polyubiquitin chains on the active-site cysteine based on single-turnover studies in the formation of diubiquitin. In addition to reconciling the topological restrictions on polyubiquitin chain assembly imposed by the standard model, Ronchi et al. (31Ronchi V.P. Klein J.M. Edwards D.J. Haas A.L. The active form of E6-associated protein (E6AP)/UBE3A ubiquitin" @default.
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