Molecular basis of Cul3 ubiquitin ligase subversion by vaccinia virus protein A55

Abstract

BTB-Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box based E3 ubiquitin ligases, which mediate protein ubiquitylation leading to proteasomal degradation. Vaccinia virus encodes three BTB-Kelch proteins, namely A55, C2 and F3. Viruses lacking A55 or C2 demonstrate altered cytopathic effect in cultured cells and altered pathology in vivo. Previous studies show that the ectromelia virus orthologue of A55, EVM150, interacts with Cul3 in cells. We show that A55 binds directly to Cul3 via its N-terminal BTB-BACK domain, and together they form a 2:2 complex in solution. The crystal structure of the A55/Cul3 complex was solved to 2.8 [A] resolution. The overall conformation and binding interfaces resemble those of the cellular BTB-BACK/Cul3 complex structures, despite low sequence similarity of A55 to cellular BTB-BACK proteins. Surprisingly, despite this structural similarity the affinity of Cul3 for A55 is significantly higher than for reported cellular BTB-BACK proteins. Detailed analysis of the binding interface suggests that I48 from A55 at the BTB/Cul3 interface is important for this high-affinity interaction and mutation at this site reduced the affinity by several orders of magnitude. I48 is conserved only in close orthologues of A55 from poxviruses, but not in C2, F3, or other poxvirus or cellular BTB-Kelch proteins. The high affinity interaction between A55 and Cul3 suggests that, in addition to directing the Cul3-RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins and thus protect substrates of these cellular adaptors from ubiquitination and degradation.