HECT-type E3 ubiquitin transferase

 

Ubiquitination is mediated by three enzymes, E1 (PDB:3cmm), E2 (PDB:1ayz) and E3 (PDB:1c4z). The first, E1, is essential for ubiquitin activation and transferring the substrate onto the second cascade enzyme, E2, which responsible for mediating repeated ubiquitination at the eventual substrate, which is brought into proximity of E2 by the active site of E3, the enzyme which also regulates substrate specificity. This entry represents the first of the three reactions occurring in this cascade: ubiquitin:[E1 ubiquitin-activating enzyme] ligase (AMP-forming)

E6AP belongs to the HECT (homologous to E6AP COOH-terminus) class of E3s, which has at least 20 members in humans. The HECT E3s are so far unique among the known classes of E3s in that they form a ubiquitin-thioester intermediate and directly catalyse substrate ubiquitination.

HECT E3s share a conserved ~40-kD COOH-terminal catalytic domain, the HECT domain (IPR000569), that has at least four biochemical activities: (i) it binds specific E2s; (ii) it accepts ubiquitin from the E2, forming a ubiquitin-thioester intermediate with its active-site cysteine; (iii) it transfers ubiquitin to the eta-amino groups of lysine side chains on the substrate by catalysing the formation of an isopeptide bond; and (iv) it transfers additional ubiquitin molecules to the growing end of the multi-ubiquitin chain. The NH2-terminal sequences of HECT E3s are not conserved and contain the primary determinants for specific substrate recognition.

HECT E3s have been implicated in cancer, hypertension, neurological disorders, and other diseases. Moreover, some pathogenic bacteria have evolved HECT-like E3s as virulence factors to manipulate host cell signalling.

 

Reference Protein and Structure

Sequences
Q05086 UniProt (2.3.2.26)
P68036 UniProt (2.3.2.23) IPR017134 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1c4z - STRUCTURE OF E6AP: INSIGHTS INTO UBIQUITINATION PATHWAY (2.6 Å) PDBe PDBsum 1c4z
Catalytic CATH Domains
3.90.1750.10 CATHdb 3.30.2410.10 CATHdb 3.10.110.10 CATHdb (see all for 1c4z)
Click To Show Structure

Enzyme Reaction (EC:2.3.2.26)

S-ubiquitinyl-[E2 ubiquitin-conjugating enzyme]-L-cysteine
CHEBI:134521ChEBI
+
lysinium residue
CHEBI:32579ChEBI
→
hydron
CHEBI:15378ChEBI
+
L-cysteine residue
CHEBI:29950ChEBI
+
N6-monoubiquitinyl-[protein]-L-lysine
CHEBI:134522ChEBI
Alternative enzyme names: HECT E3 ligase, Ubiquitin transferase HECT-E3,

Enzyme Mechanism

Introduction

Occurs in the active site of E3. Specific binding motifs are present within E3 to direct ubiquitination of lysine side chains from selected protein substrates. The presence of hydrogen bond acceptor residues enhances the nucleophilic character of the attacking lysine. Activated ubiquitin is brought into the active site by the ubiquitin loaded E2 enzyme. In the first step the enzyme transfers ubiquitin from the E2 ubiquitin-conjugating enzyme (EC 2.3.2.23) to a cysteine residue in its HECT domain (which is located in the C-terminal region), forming a thioester bond. In a subsequent step the enzyme transfers the ubiquitin to an acceptor protein, resulting in the formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and the eta-amino group of an L-lysine residue of the acceptor protein.

Catalytic Residues Roles

UniProt PDB* (1c4z)
Cys86 Cys86D The catalytic cysteine of the substrate E2. Acts as a nucleofuge. nucleofuge
Asp630 Asp607(113)A It is possible that this Asp is necessary to stabilise the L shape conformation of the HECT domain as well as to provide structural integrity to the N lobe when the C lobe is displaced. Alternatively, this Asp may be required for catalysis, although it appears unlikely to have such a role, since it is not in close proximity to the catalytic cysteine in either conformation. electrostatic stabiliser
Arg529, Glu562, Glu573, Asp630, His841 Arg506(12)A, Glu539(45)A, Glu550(56)A, Asp607(113)A, His818(324)A Identified as critical to catalysis, but exact role in the reaction mechanism not yet clear. It is likely that at least one functions as a general acid/base to activate the E3 cysteine and the final substrate protein's lysine residue. unknown
Cys843 Cys820(326)A Acts as the catalytic nucleophile that transfers the ubiquinyl group from E2 cysteine residue to the substrate protein's lysine. covalent catalysis
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Buetow L et al. (2016), Nat Rev Mol Cell Biol, 17, 626-642. Structural insights into the catalysis and regulation of E3 ubiquitin ligases. DOI:10.1038/nrm.2016.91. PMID:27485899.
  2. Zhang W et al. (2016), Mol Cell, 62, 121-136. System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes. DOI:10.1016/j.molcel.2016.02.005. PMID:26949039.
  3. Gong W et al. (2015), Acta Crystallogr F Struct Biol Commun, 71, 1251-1257. Structure of the HECT domain of human WWP2. DOI:10.1107/s2053230x1501554x. PMID:26457515.
  4. Kamadurai HB et al. (2013), Elife, 2, e00828-. Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3. DOI:10.7554/elife.00828. PMID:23936628.
  5. Meyer HJ et al. (2013), Elife, 2, e01127-. Caught in the act. DOI:10.7554/elife.01127. PMID:23936629.
  6. Maspero E et al. (2013), Nat Struct Mol Biol, 20, 696-701. Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming. DOI:10.1038/nsmb.2566. PMID:23644597.
  7. Kim HC et al. (2011), EMBO Rep, 12, 334-341. Structure and function of a HECT domain ubiquitin-binding site. DOI:10.1038/embor.2011.23. PMID:21399621.
  8. Maspero E et al. (2011), EMBO Rep, 12, 342-349. Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation. DOI:10.1038/embor.2011.21. PMID:21399620.
  9. Lin DY et al. (2011), J Biol Chem, 286, 441-449. Biochemical and Structural Studies of a HECT-like Ubiquitin Ligase from Escherichia coli O157:H7. DOI:10.1074/jbc.m110.167643. PMID:20980253.
  10. van Wijk SJ et al. (2010), FASEB J, 24, 981-993. The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. DOI:10.1096/fj.09-136259. PMID:19940261.
  11. Ye Y et al. (2009), Nat Rev Mol Cell Biol, 10, 755-764. Building ubiquitin chains: E2 enzymes at work. DOI:10.1038/nrm2780. PMID:19851334.
  12. Capili AD et al. (2007), Curr Opin Struct Biol, 17, 726-735. Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways. DOI:10.1016/j.sbi.2007.08.018. PMID:17919899.
  13. Scheffner M et al. (2007), BMC Biochem, 8, S6-. HECT E3s and human disease. DOI:10.1186/1471-2091-8-s1-s6. PMID:18047743.
  14. Ogunjimi AA et al. (2005), Mol Cell, 19, 297-308. Regulation of Smurf2 Ubiquitin Ligase Activity by Anchoring the E2 to the HECT Domain. DOI:10.1016/j.molcel.2005.06.028. PMID:16061177.
  15. Passmore LA et al. (2004), Biochem J, 379, 513-525. Getting into position: the catalytic mechanisms of protein ubiquitylation. DOI:10.1042/bj20040198. PMID:14998368.
  16. Verdecia MA et al. (2003), Mol Cell, 11, 249-259. Conformational Flexibility Underlies Ubiquitin Ligation Mediated by the WWP1 HECT Domain E3 Ligase. DOI:10.1016/s1097-2765(02)00774-8.
  17. Huang L et al. (1999), Science, 286, 1321-1326. Structure of an E6AP-UbcH7 Complex: Insights into Ubiquitination by the E2-E3 Enzyme Cascade. DOI:10.1126/science.286.5443.1321. PMID:10558980.
  18. Scheffner M et al. (1995), Nature, 373, 81-83. Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade. DOI:10.1038/373081a0. PMID:7800044.

Step 1.

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Catalytic Residues Roles

Residue Roles
Cys86D nucleofuge
Cys820(326)A covalent catalysis
Glu539(45)A unknown
Glu550(56)A unknown
Asp607(113)A electrostatic stabiliser
Arg506(12)A unknown

Chemical Components

Step 1.

Contributors

Nozomi Nagano, Gemma L. Holliday, Sophie T. Williams