REFINED CRYSTAL STRUCTURES OF TNF-ALPHA AND TNF MUTANT R31D. C. Reed*, Z.-Q. Fu*, J. Wu*, Y.-N. Xue, M.-J. Chen and I.T. Weber*. *Department of Pharmacology, Department of Microbiology and Immunology, Jefferson Cancer Center, Thomas Jefferson University, Philadelphia PA 19107, USA.

Crystal structures have been determined of recombinant human tumor necrosis factor-( (TNF-() and its R31D mutant that preferentially binds to TNF receptor R1 with five times greater affinity than to receptor R2. Crystals of the wild type TNF were of space group P41212 and had unit cell dimensions of a=b=94.7 and c=117.4 Å. Refinement of the structure gave an R-factor of 22.3% at 2.5 Å resolution. The crystals of TNF R31D mutant diffracted to 2.3Å resolution, and were of identical space group to the wild type with unit cell dimensions of a=b=95.4 and c=116.2 Å, and the structure was refined to an R-factor of 21.8%. Almost continuous electron density was observed throughout both structures, although the first five residues of the N-termini appear to be disordered. Comparison of the structures of the wild type and mutant TNF showed that the two trimers were similar with an rms deviation of 0.77 Å for main chain atoms, however, the subunits within each trimer were more variable with rms deviations of over 1.05 Å for pairwise comparison of main chain atoms. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. The Arg 31 of wild type TNF is predicted to form an ionic interaction with an identical glutamic acid in both receptors R1 and R2. In the TNF R31D mutant, modeling suggested that this interaction is replaced by interaction with a histidine in R1, but there is no equivalent interaction in R2, consistent with the observed greater affinity of the R31D mutant for receptor R1 compared to R2.