Structural insights into non-covalent ubiquitin activation of the cIAP1-UbcH5B∼ubiquitin complex

Abstract

Ubiquitin (Ub)-conjugating enzymes and Ub ligases control protein degradation and regulate many cellular processes in eukaryotes. Cellular inhibitor of apoptosis protein-1 (cIAP1) plays a central role in apoptosis and tumor necrosis factor signaling. It harbors a C-terminal RING domain that homodimerizes to recruit E2∼Ub (where ∼ denotes a thioester bond) complex to catalyze Ub transfer. Noncovalent Ub binding to the backside of the E2 Ub-conjugating enzyme UbcH5 has previously been shown to enhance RING domain activity, but the molecular basis for this enhancement is unclear. To investigate how dimeric cIAP1 RING activates E2∼Ub for Ub transfer and what role noncovalently bound Ub has in Ub transfer, here we determined the crystal structure of the cIAP1 RING dimer bound to both UbcH5B covalently linked to Ub (UbcH5B–Ub) and a noncovalent Ub to 1.7 Å resolution. The structure along with biochemical analyses revealed that the cIAP1 RING domain interacts with UbcH5B–Ub and thereby promotes the formation of a closed UbcH5B–Ub conformation that primes the thioester bond for Ub transfer. We observed that the noncovalent Ub binds to the backside of UbcH5B and abuts UbcH5B's α1β1-loop, which, in turn, stabilizes the closed UbcH5B–Ub conformation. Our results disclose the mechanism by which cIAP1 RING dimer activates UbcH5B∼Ub and indicate that noncovalent Ub binding further stabilizes the cIAP1-UbcH5B∼Ub complex in the active conformation to stimulate Ub transfer.

Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34

“…Detailed analysis of the Cdc34-Ub structure reveals that CTD prox engages in an extensive network of intramolecular interactions with the backside of Cdc34 that is known to bind non-covalent Ub to allosterically enhance activity in a subset of E2s 59 (Fig. 6b and Supplementary Fig.…”

Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site

“…Noncovalent Ub binding can influence targeting, processivity, and rates of ubiquitylation reactions. As examples, noncovalent Ub binding to the E2 UBE2D2 allosterically modulates binding to partner RING domain E3s (19,20), while noncovalent binding to HECT E3s in the NEDD4 family can modulate catalytic activity and/or processivity of substrate ubiquitylation (21). The first RING domain discovered to possess a Ub-binding exosite was APC11, a subunit of one of the most complicated and unusual E3 ligases, the anaphase-promoting complex/cyclosome (APC/C) (22).…”

Structural basis of generic versus specific E2–RING E3 interactions in protein ubiquitination

“…(a) Ribbon representation of a single RNF4 monomer from UBE2D1~Ub:RNF4 complex (PDB: 4AP4) showing the zinc ions as spheres, the residues involved in zinc‐coordination and the allosteric linchpin residue as ball‐and‐sticks, and demarcating the E2‐binding site. The ribbon diagram is colored to reflect the average pairwise positional shift of each overlapping C α atom between RNF4 and unique RING cores from all available E2–RING E3 complex structures including RNF146 (PDB: 4QPL), TRIM25 (PDB: 5FER), BIRC2 (PDB: 6HPR), BIRC3 (PDB: 3 EB6), BIRC7 (PDB: 4AUQ), RNF38 (PDB: 4V3K), RNF25 (PDB: 5D1M), RNF165 (PDB: 5D0M), MDM2 (PDB: 5MNJ), TRIM23 (PDB: 5VZW), RNF13 (PDB: 5ZBU), E4B (PDB: 3L1Z), RNF2 (PDB: 3RPG), GP78 (PDB: 2LXP), SIZ1 (PDB: 5JNE), c‐CBL (PDB: 1FBV), RBX1 (PDB: 4P5O), TRAF6 (PDB: 3HCT), TRIM5 (PDB: 4TKP), RNF8 (PDB: 4WHV), ZNRF1 (PDB: 5YWR), LNX1 (PDB: 5H7S), CHIP (PDB: 2C2V), and FANCL (PDB: 2CCG) . (b) UBE2D1~Ub:RNF4 complex highlighting position of the RING domain relative to the E2, with the allosteric linchpin residue coordinating E2 as well as the donor Ub shown as ball‐and‐sticks.…”

“…However, an increasing number of RING domains have been found to be functional only when oligomeric. For example, RNF4 and BIRC‐family RING E3s form homodimers via their RING domains . Similarly, some RING domains that lack intrinsic E2~Ub binding and E3 ligase activity, such as polycomb BMI1 and double minute protein (MDMX), heterodimerize with their respective active, RING domain‐containing partners to become functional .…”

“…For example, RNF4 and BIRC-family RING E3s form homodimers via their RING domains. [49][50][51][52] Similarly, some RING domains that lack intrinsic E2~Ub binding and E3 ligase activity, such as polycomb BMI1 and double minute protein (MDMX), heterodimerize with their respective active, RING domaincontaining partners to become functional. [53][54][55] Regulation of RING activity by homo-or heterodimerization has recently been reviewed in further detail elsewhere.…”