Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells [Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690]. Here we describe the structure of Apo2L at 1.3 A resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.

Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.

Formation of a complex between Apo2L (also called TRAIL) and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains. We report the crystal structure of the complex between Apo2L and the ectodomain of DR5. The structure shows three elongated receptors snuggled into long crevices between pairs of monomers of the homotrimeric ligand. The interface is divided into two distinct patches, one near the bottom of the complex close to the receptor cell surface and one near the top. Both patches contain residues that are critical for high-affinity binding. A comparison to the structure of the lymphotoxin-receptor complex suggests general principles of binding and specificity for ligand recognition in the TNF receptor superfamily.

We have probed the relative contribution of polar and nonpolar interactions to antibody-antigen interaction by measuring the effect of single amino acid substitutions in an humanized anti-p185HER2 antibody (hu4D5-5) on the thermodynamics of antigen binding. First we mapped the functional epitope by complete alanine-scan mutagenesis of the antibody complementarity-determining region (CDR). Four residues, H91 in VL and R50, W95, and Y100a in VH, make large contributions to the free energy of binding (delta delta G > 3 kcal mol-1) and have delta delta G > delta delta H. These residues are clustered in a shallow pocket on the antibody surface in the X-ray structure determined for hu4D5 Fv. The majority of other CDR residues make less energetically important contributions (delta delta G < 1 kcal mol-1) to binding but have delta delta H > delta delta G, suggesting that the wild-type side chain does contact antigen but the loss in entropy, perhaps through restriction of side-chain conformational freedom, offsets the favorable enthalpic term. Effects of Phe and Ala substitutions on the delta G and delta Cp of antigen binding indicate that the favorable contribution of antibody tyrosine residues to binding results primarily from burial of the aromatic ring in the interface with antigen. Burial of the phenyl ring has a favorable delta H at 25 degrees C but at least for one site (VL-Y92) is opposed by delta S. This latter feature is inconsistent with the thermodynamics predicted for the hydrophobic effect based on hydrocarbon-transfer experiments.(ABSTRACT TRUNCATED AT 250 WORDS)