Characterization of RING-Between-RING E3 Ubiquitin Transfer Mechanisms

  • Katherine H. Reiter
  • Rachel E. KlevitEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)


Protein ubiquitination is an essential posttranslational modification that regulates nearly all cellular processes. E3 ligases catalyze the final transfer of ubiquitin (Ub) onto substrates and thus are important temporal regulators of ubiquitin modifications in the cell. E3s are classified by their distinct transfer mechanisms. RING E3s act as scaffolds to facilitate the transfer of Ub from E2-conjugating enzymes directly onto substrates, while HECT E3s form an E3~Ub thioester intermediate prior to Ub transfer. A third class, RING-Between-RING (RBR) E3s, are classified as RING/HECT hybrids based on their ability to engage the E2~Ub conjugate via a RING1 domain while subsequently forming an obligate E3~Ub intermediate prior to substrate modification. RBRs comprise the smallest class of E3s, consisting of only 14 family members in humans, yet their dysfunction has been associated with neurodegenerative diseases, susceptibility to infection, inflammation, and cancer. Additionally, their activity is suppressed by auto-inhibitory domains that block their catalytic activity, suggesting their regulation has important cellular consequences. Here, we identify technical hurdles faced in studying RBR E3s and provide protocols and guidelines to overcome these challenges.

Key words

Ubiquitin Transthiolation Ubiquitin E3 ligase RBR HHARI Parkin HOIP RING-Between-RING 


  1. 1.
    Wenzel DM, Lissounov A, Brzovic PS, Klevit RE (2011) UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474(7349):105–108. Scholar
  2. 2.
    Kelsall IR, Duda DM, Olszewski JL, Hofmann K, Knebel A, Langevin F, Wood N, Wightman M, Schulman BA, Alpi AF (2013) TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes. EMBO J 32(21):2848–2860. Scholar
  3. 3.
    Ho SR, Mahanic CS, Lee YJ, Lin WC (2014) RNF144A, an E3 ubiquitin ligase for DNA-PKcs, promotes apoptosis during DNA damage. Proc Natl Acad Sci U S A 111(26):E2646–E2655. Scholar
  4. 4.
    Smit JJ, Monteferrario D, Noordermeer SM, van Dijk WJ, van der Reijden BA, Sixma TK (2012) The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J 31(19):3833–3844. Scholar
  5. 5.
    Stieglitz B, Morris-Davies AC, Koliopoulos MG, Christodoulou E, Rittinger K (2012) LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep 13(9):840–846. Scholar
  6. 6.
    Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392(6676):605–608. Scholar
  7. 7.
    Dove KK, Kemp HA, Di Bona KR, Reiter KH, Milburn LJ, Camacho D, Fay DS, Miller DL, Klevit RE (2017) Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development. Proc Natl Acad Sci U S A 114(32):E6576–E6584. Scholar
  8. 8.
    Qiu X, Fay DS (2006) ARI-1, an RBR family ubiquitin-ligase, functions with UBC-18 to regulate pharyngeal development in C. elegans. Dev Biol 291(2):239–252. Scholar
  9. 9.
    Iwai K, Fujita H, Sasaki Y (2014) Linear ubiquitin chains: NF-kappaB signalling, cell death and beyond. Nat Rev Mol Cell Biol 15(8):503–508. Scholar
  10. 10.
    Ikeda F (2015) Linear ubiquitination signals in adaptive immune responses. Immunol Rev 266(1):222–236. Scholar
  11. 11.
    Kumar A, Aguirre JD, Condos TE, Martinez-Torres RJ, Chaugule VK, Toth R, Sundaramoorthy R, Mercier P, Knebel A, Spratt DE, Barber KR, Shaw GS, Walden H (2015) Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis. EMBO J 34(20):2506–2521. Scholar
  12. 12.
    Riley BE, Lougheed JC, Callaway K, Velasquez M, Brecht E, Nguyen L, Shaler T, Walker D, Yang Y, Regnstrom K, Diep L, Zhang Z, Chiou S, Bova M, Artis DR, Yao N, Baker J, Yednock T, Johnston JA (2013) Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases. Nat Commun 4:1982. Scholar
  13. 13.
    Trempe JF, Sauve V, Grenier K, Seirafi M, Tang MY, Menade M, Al-Abdul-Wahid S, Krett J, Wong K, Kozlov G, Nagar B, Fon EA, Gehring K (2013) Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science 340(6139):1451–1455. Scholar
  14. 14.
    Wauer T, Komander D (2013) Structure of the human Parkin ligase domain in an autoinhibited state. EMBO J 32(15):2099–2112. Scholar
  15. 15.
    Dove KK, Olszewski JL, Martino L, Duda DM, Wu XS, Miller DJ, Reiter KH, Rittinger K, Schulman BA, Klevit RE (2017) Structural studies of HHARI/UbcH7 approximately Ub reveal unique E2 approximately Ub conformational restriction by RBR RING1. Structure 25(6):890–900.e5. Scholar
  16. 16.
    Duda DM, Olszewski JL, Schuermann JP, Kurinov I, Miller DJ, Nourse A, Alpi AF, Schulman BA (2013) Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism. Structure 21(6):1030–1041. Scholar
  17. 17.
    Yuan L, Lv Z, Atkison JH, Olsen SK (2017) Structural insights into the mechanism and E2 specificity of the RBR E3 ubiquitin ligase HHARI. Nat Commun 8(1):211. Scholar
  18. 18.
    Stieglitz B, Rana RR, Koliopoulos MG, Morris-Davies AC, Schaeffer V, Christodoulou E, Howell S, Brown NR, Dikic I, Rittinger K (2013) Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP. Nature 503(7476):422–426. Scholar
  19. 19.
    Lechtenberg BC, Rajput A, Sanishvili R, Dobaczewska MK, Ware CF, Mace PD, Riedl SJ (2016) Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation. Nature 529(7587):546–550. Scholar
  20. 20.
    Dove KK, Klevit RE (2017) RING-between-RING E3s ligases: emerging themes amid the variations. J Mol Biol 429:3363. Scholar
  21. 21.
    Chaugule VK, Burchell L, Barber KR, Sidhu A, Leslie SJ, Shaw GS, Walden H (2011) Autoregulation of Parkin activity through its ubiquitin-like domain. EMBO J 30(14):2853–2867. Scholar
  22. 22.
    Scott DC, Rhee DY, Duda DM, Kelsall IR, Olszewski JL, Paulo JA, de Jong A, Ovaa H, Alpi AF, Harper JW, Schulman BA (2016) Two distinct types of E3 ligases work in unison to regulate substrate Ubiquitylation. Cell 166(5):1198–1214.e24. Scholar
  23. 23.
    Pickrell AM, Youle RJ (2015) The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease. Neuron 85(2):257–273. Scholar
  24. 24.
    Eiyama A, Okamoto K (2015) PINK1/Parkin-mediated mitophagy in mammalian cells. Curr Opin Cell Biol 33:95–101. Scholar
  25. 25.
    Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, Gourlay R, Burchell L, Walden H, Macartney TJ, Deak M, Knebel A, Alessi DR, Muqit MM (2012) PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating serine 65. Open Biol 2(5):120080. Scholar
  26. 26.
    Shiba-Fukushima K, Imai Y, Yoshida S, Ishihama Y, Kanao T, Sato S, Hattori N (2012) PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy. Sci Rep 2:1002. Scholar
  27. 27.
    Kane LA, Lazarou M, Fogel AI, Li Y, Yamano K, Sarraf SA, Banerjee S, Youle RJ (2014) PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. J Cell Biol 205(2):143–153. Scholar
  28. 28.
    Kazlauskaite A, Kondapalli C, Gourlay R, Campbell DG, Ritorto MS, Hofmann K, Alessi DR, Knebel A, Trost M, Muqit MM (2014) Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem J 460(1):127–139. Scholar
  29. 29.
    Koyano F, Okatsu K, Kosako H, Tamura Y, Go E, Kimura M, Kimura Y, Tsuchiya H, Yoshihara H, Hirokawa T, Endo T, Fon EA, Trempe JF, Saeki Y, Tanaka K, Matsuda N (2014) Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510(7503):162–166. Scholar
  30. 30.
    Wauer T, Simicek M, Schubert A, Komander D (2015) Mechanism of phospho-ubiquitin-induced PARKIN activation. Nature 524(7565):370–374. Scholar
  31. 31.
    Ikeda F, Deribe YL, Skanland SS, Stieglitz B, Grabbe C, Franz-Wachtel M, van Wijk SJ, Goswami P, Nagy V, Terzic J, Tokunaga F, Androulidaki A, Nakagawa T, Pasparakis M, Iwai K, Sundberg JP, Schaefer L, Rittinger K, Macek B, Dikic I (2011) SHARPIN forms a linear ubiquitin ligase complex regulating NF-kappaB activity and apoptosis. Nature 471(7340):637–641. Scholar
  32. 32.
    Gerlach B, Cordier SM, Schmukle AC, Emmerich CH, Rieser E, Haas TL, Webb AI, Rickard JA, Anderton H, Wong WW, Nachbur U, Gangoda L, Warnken U, Purcell AW, Silke J, Walczak H (2011) Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471(7340):591–596. Scholar
  33. 33.
    Tokunaga F, Nakagawa T, Nakahara M, Saeki Y, Taniguchi M, Sakata S, Tanaka K, Nakano H, Iwai K (2011) SHARPIN is a component of the NF-kappaB-activating linear ubiquitin chain assembly complex. Nature 471(7340):633–636. Scholar
  34. 34.
    Yagi H, Ishimoto K, Hiromoto T, Fujita H, Mizushima T, Uekusa Y, Yagi-Utsumi M, Kurimoto E, Noda M, Uchiyama S, Tokunaga F, Iwai K, Kato K (2012) A non-canonical UBA-UBL interaction forms the linear-ubiquitin-chain assembly complex. EMBO Rep 13(5):462–468. Scholar
  35. 35.
    Kirisako T, Kamei K, Murata S, Kato M, Fukumoto H, Kanie M, Sano S, Tokunaga F, Tanaka K, Iwai K (2006) A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J 25(20):4877–4887. Scholar
  36. 36.
    Pruneda JN, Littlefield PJ, Soss SE, Nordquist KA, Chazin WJ, Brzovic PS, Klevit RE (2012) Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases. Mol Cell 47(6):933–942. Scholar
  37. 37.
    Dove KK, Stieglitz B, Duncan ED, Rittinger K, Klevit RE (2016) Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms. EMBO Rep 17(8):1221–1235. Scholar
  38. 38.
    Ordureau A, Sarraf SA, Duda DM, Heo JM, Jedrychowski MP, Sviderskiy VO, Olszewski JL, Koerber JT, Xie T, Beausoleil SA, Wells JA, Gygi SP, Schulman BA, Harper JW (2014) Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol Cell 56(3):360–375. Scholar
  39. 39.
    Tokunaga F, Sakata S, Saeki Y, Satomi Y, Kirisako T, Kamei K, Nakagawa T, Kato M, Murata S, Yamaoka S, Yamamoto M, Akira S, Takao T, Tanaka K, Iwai K (2009) Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation. Nat Cell Biol 11(2):123–132. Scholar
  40. 40.
    Sarraf SA, Raman M, Guarani-Pereira V, Sowa ME, Huttlin EL, Gygi SP, Harper JW (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496(7445):372–376. Scholar
  41. 41.
    Borodovsky A, Ovaa H, Kolli N, Gan-Erdene T, Wilkinson KD, Ploegh HL, Kessler BM (2002) Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem Biol 9(10):1149–1159CrossRefGoogle Scholar
  42. 42.
    Park S, Foote PK, Krist DT, Rice SE, Statsyuk AV (2017) UbMES and UbFluor: novel probes for RBR E3 ubiquitin ligase PARKIN. J Biol Chem 292:16539. Scholar
  43. 43.
    Christensen DE, Brzovic PS, Klevit RE (2007) E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol 14(10):941–948. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of WashingtonSeattleUSA

Personalised recommendations