IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

Our SILAC data indicated that IFIT2 and IFIT3 were enriched to a similar degree in IFIT1-bound samples (Figure 1 ), suggesting that a heterocomplex of IFIT2:IFIT3 was the more likely form to interact with IFIT1 in the cell. This prompted us to investigate the IFIT2:IFIT3 interaction in more detail. The delayed elution of the dimeric IFIT2 from the size exclusion column fortuitously enabled us to analyse the IFIT2:IFIT3 interaction by SEC-MALS. Although no interaction between these two proteins was detected when incubated at 4 C, we observed the appearance of a heterodimeric complex when these proteins were incubated at 37 C ( Figure 2B ) indicating that addition of energy was required for this heterocomplex to form. The crystal structure of IFIT2 revealed that dimerisation of the protein was due to a domain swap of three -helices that constitute one and a half TPRs in the N-terminal domain (7) (Figure S6A and shown schematically in Figure 6 ). Nucleic acid binding activity of IFIT2 was localised to the dimer interface surfaces of the C-terminal domain that form a large positively charged pocket ( Figure S6B ). The electrostatic surface potential of an IFIT3 molecular model, based on the IFIT2 crystal structure, is shown in Figure S6C . The IFIT3 model lacks a positively-charged nucleic acid binding surface like that of IFIT2.