VIAF is a member of a conserved protein family that associates with animal IAPs (inhibitor of apoptosis proteins). VIAF itself substantially protects cells from Fas- and Bax-induced apoptosis, while coexpression of VIAF with suboptimal quantities of XIAP confers almost complete protection from these inducers. VIAF and XIAP activate JNK in a synergistic manner. Hence, VIAF is a novel cofactor which modulates the anti-apoptotic and signaling properties of the IAP family. In order to obtain a basis for understanding the function of VIAF at the molecular level, we have initiated a determination of the three-dimensional structure of VIAF using NMR spectroscopy. Full length VIAF contains 239 residues, but two?hybrid-screening studies have shown that the C-terminal 128 residue region of the protein is necessary and sufficient for interaction with Op-IAP. Hence we have expressed the C-terminal domain of VIAF, henceforth referred to as VIAFC in E..coli using the pET11 vector. Cultures were grown in minimal media containing either 15N or 15N plus 13C labeled ammonium chloride and glucose, respectively. The protein was purified from inclusion bodies and successfully refolded. NMR spectra were recorded on the protein dissolved in a 10 mM phosphate buffer, pH 7.4, at 35 degrees C. In the process of obtaining NMR backbone and sidechain signal assignments, a novel constant-time HSQC approach was developed to assign sidechain aromatic signals. A moderate-resolution structure of VIAFC, determined using over 1000 NOE constraints, has been obtained and shows that its three dimensional structure is surprisingly similar to that of phosducin, a protein of limited sequence homology. Five sets of different heteronuclear residual dipolar couplings have been measured, and are being used to refine and validate the structure determined using NOEs and coupling constants. After the three dimensional solution structure of VIAFC is completed, the region of VIAFC that interacts with XIAP will be determined.
