PDBsum entry 5d1k

Ligase

PDB id: 5d1k

Contents

Protein chains

146 a.a.

110 a.a.

Ligands

EDO ×3

PEG

OXL

Metals

_ZN ×2

Waters ×272

References listed in PDB file

Key reference

• Title: Insights into ubiquitination from the unique clamp-Like binding of the ring e3 ao7 to the e2 ubch5b.
• Authors: S.Li, Y.H.Liang, J.Mariano, M.B.Metzger, D.K.Stringer, V.A.Hristova, J.Li, P.A.Randazzo, Y.C.Tsai, X.Ji, A.M.Weissman.
• Ref.: J Biol Chem, 2015, 290, 30225-30239. [DOI no: 10.1074/jbc.M115.685867]
• PubMed id: 26475854

Abstract

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.