We favor the interpretation that removal of helix 1 destabilizes the HECT domain to produce a more relaxed version of the enzyme that exhibits greater intradomain flexibility. (13). An enzymatic cascade consisting of Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), and Ub ligase (E3) is responsible for catalyzing Ub conjugation to target proteins. The E1 enzyme activates Ub for transfer by adenylating its C terminus in an ATP-dependent step. Ub is then transferred to the E1 active site cysteine, forming a thioester bond between E1 and Ub. The activated Ub is transferred to E2 enzyme in a transthiolation reaction and is conjugated to target proteins by the action of E3 Ub ligases (3). The Lenampicillin hydrochloride E2 and E3 enzymes provide substrate specificity to the Ub conjugation reaction (4). Approximately 600 E3s exist in the human proteome, 28 of which belong to the HECT (homologous to E6AP C terminus) domain family (5). HECT domain E3s possess a 350-amino acid C-terminal HECT domain containing a conserved catalytic cysteine that participates in the transfer of Ub to substrate. Mutation, abnormal expression, or misregulation of these enzymes predisposes to the development of cancer and disease (1,6). Structural studies describe the canonical HECT domain architecture and provide insight into its mechanism of catalysis (79). HECT domains are composed of two subdomains connected by a flexible peptide linker. The N-terminal (N) lobe contains the E2 binding region, and the C-terminal (C) lobe contains the catalytic cysteine. Lenampicillin hydrochloride In the structure of the E6AP-UbcH7 complex (Protein Data Bank codes 1C4Z and 1D5F), the catalytic cysteines of E2 and E3 are separated by 41 , suggesting that a substantial conformational rearrangement is required to achieve Ub transfer (7). Analysis of the structure of the WWP1 HECT domain (PDB code 1ND7) partially addresses how Ub is transferred from the E2 to the E3 catalytic cysteine by illustrating conformational flexibility of the HECT domain (9). In the WWP1 structure modeled in a complex with UbcH5, Itgax the C-lobe is rotated about the hinge region, placing Lenampicillin hydrochloride it in closer proximity to the E2 cysteine and closing the distance between active site cysteines to 16 . Mutations in this hinge loop that restrict HECT domain rotation decrease activity (9). Additional structural elements within the HECT domain that modulate conformation or activity remain unknown. HUWE1 (also called ARF-BP1, Mule, Lasu1, Ureb1, E3 histone, and HectH9) is a 482-kDa HECT domain E3 Ub ligase implicated in the regulation of cell proliferation, apoptosis, DNA damage response, and base excision repair (1016). We recovered this enzyme in immunoprecipitations using Ub C-terminal electrophilic probes (17). After an initial biochemical characterization (17), we completed a structural and biophysical analysis of the HECT domain to understand modulation of its robustin vitroactivity. Here we present crystal structures of the HUWE1 HECT domain and characterize a structural element that both stabilizes this domain and modulates its activity. This structural element, the 1 helix, is an important component of the HECT domain that largely restricts its autoubiquitination Lenampicillin hydrochloride activity while only nominally affecting Mcl-1 ubiquitination activity. == EXPERIMENTAL PROCEDURES == == == == == == Plasmids == HECT domain constructs of HUWE1 (amino acids 39934374 or amino acids 40124374) were cloned into a modified pET-28a plasmid (Novagen) containing a human rhinovirus 3C (HRV3C) protease site to generate an N-terminal His6fusion protein for use in biochemical assays. The mutants C4341A and C4099A/C4184A/C4367A were generated using Lenampicillin hydrochloride site-directed mutagenesis (Stratagene). For biochemical assays with radiolabeled substrate, FLAG-Mcl-1 (amino acids 1327) and Ub were both cloned with an N-terminal protein kinase A site for32P labeling into pET-16b and pET-28a with the HRV3C site, respectively, as previously reported (17). UBE2L3 was cloned into the pET-28a plasmid (Novagen). == Bacterial Protein Expression and Purification.
