Ubiquitin ligase(E6AP)/ubiquitin-conjugating enzyme(UbcH7) complex
Homo sapiens (human)
Proteins destined for secretion, the plasma membrane or the cell surface are modified and refolded in the endoplasmic reticulum (ER). The correctly folded proteins are transferred to the secretion pathway of the Golgi body. During this process, proteins that fail to fold correctly are identified by a quality control procedure and subjected to ER-associated degradation (ERAD). These proteins are retranslocated across the ER membrane into the cytoplasm and degraded by the 26S proteasome. Target proteins are polyubiquitilated in the ER, and this serves as a marker "tag". Ubiquitin is a small protein with 76 amino acids. Beyond this quality control function the ubiquitin-proteasome system also plays a crucial role in cell cycle, apoptosis, metabolic control, signaling, transcriptional regulation, stress response and immunologic response. Ubiquitin is bound in an isopeptide linkage through its C-terminal glycine to the N-terminus or to the epsilon amino group of internal lysine residues of target proteins. This linkage is formed by the multyenzyme system composed of ubiquitin-activating enzyme (E1, Uba), ubiquitin-conjugating enzyme (E2, Ubc) and ubiquitin-protein ligase (E3, Ubl). Firstly, ubiquitin is activated by ATP-dependent trans-esterification and bound to a cysteine residue of an E1 enzyme. Next, ubiquitin is transferred to a cystein residue of an E2 enzyme and finally linked to the substrate by an E3 enzyme. Polyubiquitination is accomplished by attaching additional ubiqutines, usually to Lys48 of the preceding ubiquitin.
Here is the crystal structure of the complex of ubiquitin-protein ligase, E6AP and the human ubiquitin-conjugating enzyme UbcH7. This is the first structure of an E3 enzyme and of an E2-E3 complex. E6AP mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The complex has a U-shaped structure with the E6AP representing the base of the N-terminal region and one side of the C-terminal region. The catalytic residue Cys820 which forms a thioester bond with ubiquitin is located in the broad cleft at the junction between the N-terminal and the C-terminal regions. The N-terminal portion of the cleft contains mostly polar and charged residues and has an overall negative charge. The C-terminal portion of the cleft contains residues that are partially exposed to solvent. Residues contributing to these features are generally conserved within the hect family to which E6AP belongs. The structure of UbcH7 is similar to that of other E2 enzymes. UbcH7 binds in a hydrophobic groove on the N-terminal E6AP using loops at one end of its beta sheet and a portion of its N-terminal alpha helix. A phenylalanine (Phe67) conserved only in the hect-specific E2 subfamily bind in the center of the hydrophobic groove of E6AP. The overall structure of E6AP does not change upon UbcH7 binding. The active-site cysteine side chains of E6AP (Cys820) and UbcH7 (Cys86) are 41angstrom apart.
Protein Data Bank (PDB)
Huang, 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) 286:1321-1326 PubMed:10558980.
author: Tomoki Matsuda