DOI: 10.1101/461806Nov 5, 2018Paper

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

BioRxiv : the Preprint Server for Biology
Chen GaoStephen C Graham

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...Continue Reading

Methods Mentioned

BETA
thermal shift
PCR
gel filtration
light scattering
transfection
GTPases
co-immunoprecipitation
thermal
Isothermal titration calorimetry
co-IP

Software Mentioned

BUSTER
GraphPad
DIALS
ALINE
Molprobity
PHENIX PHASER - MR
STARANISO
xia2
COOT
CCP4

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