Fig. 4

The TRAF RING dimer interface is conserved allowing formation of TRAF heterodimers. a Conservation surface mapping of the RING and first three ZFs of TRAFs 2, 3, 5 and 6 mapped on the structure of TRAF6 (PDB: 3HCS) using Chimera⁵¹. Magenta represents identical residues, while cyan shows low levels of similarity. The interacting RZ₃ molecule is shown in ribbon format (grey). Zinc atoms are shown as spheres, while coordinating side chains are shown as sticks with oxygen, nitrogen and sulphur atoms in red, blue and yellow, respectively. b Sequence alignment with identical colouring as on surface in panel a. Residues important for dimerisation are indicated by red asterisks, zinc-coordinating residues by black boxes, and the Arg residues at the ubiquitin binding interface are indicated by arrows. Domains are shown as grey boxes, and the linker-helix is indicated above the sequence. c Analytical size exclusion profile of TRAF6, TRAF5 and a mixture of the two. Equivalent fractions from each run were resolved by 16% SDS-PAGE and stained with Coomassie Blue. d Top: discharge of fluorescently labelled Ubc13~Ub thioester conjugate following addition of TRAF6, TRAF5, mutants of each, and mixtures of these proteins as indicated. Bottom: multi-turnover assays of the same TRAF proteins using fluorescently labelled ubiquitin