Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~ubiquitin conformation

Funding: Investigator Award from the Wellcome Trust (098391/Z/12/Z) and (217196/Z/19/Z) and a Programme grant from Cancer Research UK (C434/A21747) to R.T.H.; J.C.P. thanks the University of St Andrews for financial support.Based on extensive structural analysis it was proposed that RING E3 ligases prime the E2~ubiquitin conjugate (E2~Ub) for catalysis by locking it into a closed conformation, where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer has yet to be experimentally tested. To test this hypothesis we use single molecule Förster Resonance Energy Transfer (smFRET) to measure the conformation of a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. We describe a real-time FRET assay with a thioester linked E2~Ub conjugate to monitor single ubiquitination events and demonstrate that ubiquitin is transferred to substrate from the closed conformation. These findings are likely to be relevant to all RING E3 catalysed reactions ligating ubiquitin and other ubiquitin-like proteins (Ubls) to substrates.Publisher PDFPeer reviewe

Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming

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1H, 13C and 15N Resonance Assignments for the Human E2 Conjugating Enzyme, UbcH7

UbcH7 is a human E2 conjugating enzyme in the ubiquitin-dependent protein degradation pathway. The resonance assignments of UbcH7 will assist in elucidating the structural basis of interactions that occur within ubiquitination

The Structure of the UbcH8−Ubiquitin Complex Shows a Unique Ubiquitin Interaction Site

Ubiquitin-mediated proteolysis utilizes a series of three key enzymes (E1, E2, and E3) to transfer and then covalently modify a substrate with ubiquitin. E2 conjugating enzymes are central proteins in this pathway responsible for the acceptance of a ubiquitin from the E1 enzyme and association with an E3 protein. All E2 enzymes covalently bind ubiquitin through a thiolester linkage between a conserved active-site cysteine on E2 and the C-terminal glycine on ubiquitin. It is not known whether E2 enzymes utilize similar surfaces and residues to coordinate a ubiquitin molecule and how this might contribute to any substrate specificity. In this work, we determined the structure of the human E2 enzyme UbcH8 (UBE2L6) covalently bound to ubiquitin by NMR spectroscopy. A disulfide bond mimicking the short-lived thiolester was formed between the two proteins providing a stable complex. Overall, the structure of UbcH8 does not undergo a significant conformational change upon forming a complex with ubiquitin. Chemical shift perturbation and cross-saturation experiments were used to identify contacts between UbcH8 and ubiquitin and those contacts used as inputs for HADDOCK molecular docking to produce the structure of the UbcH8-ubiquitin complex. An ensemble of 16 structures (root-mean-square deviation of 0.83 A) showed that ubiquitin interacts with the linker region prior to the alpha5 helix as well as residues near the catalytic site. This region corresponds to an area of negative potential on the UbcH8 surface and is considerably different from other E2-ubiquitin interaction sites. Our findings indicate the positioning of ubiquitin on UbcH8 would still allow interaction with E1 and E3 enzymes. Together, the results suggest the UbcH8-ubiquitin complex may provide an additional level of specificity in the ubiquitination pathway