CRYSTAL STRUCTURE ANALYSIS OF HUMAN TRANSTHYRETIN COMPLEXES WITH FLUORESCENT PROBES. Vivian Cody, Joseph R. Luft, Walter Pangborn, Hauptman-Woodward Medical Research Institute, Inc., Buffalo, NY 14203

Fluorescent probes of the N-arylaminonaphthalene sulfonate type are used to assess the hydrophobicity of protein binding sites and as a means of monitoring conformational changes in biological macromolecules. Structure-activity data show that they can also act as competitive inhibitors for thyroxine (T₄) binding to transthyretin (TTR). Fluorescence quenching studies of 8-anilino-1-naphthalene sulfonic acid (ANS) by competitive displacement of T₄ from TTR was used to determine the binding affinity of T₄ and to describe negative cooperativity in binding the hormone to the two equivalent sites on the TTR tetramer. These data supported two theoretical models for ANS quenching - one showing independent actions of the two hormone binding sites, and the other requiring interaction between the two sites. Similarly, the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-AEDANS) was shown to form a covalent bond with Cys-10 of TTR. In order to better understand negative cooperativity in hormone binding to TTR, we have carried out the X-ray crystal structure determination of human TTR co-crystallized with various fluorescent probes and report structural results for TTR complexed with ANS and 1,8-AEDANS. Both crystals diffract to 1.9Å resolution and crystallize in the orthorhombic space group P2₁2₁2 with two independent monomers in the asymmetric unit. Cell dimensions are isomorphous to previously reported lattices. Refinement of each structure was carried out to 1.9Å resolution without inhibitor contributions using the program PROLSQ. Difference (F_o-F_c) electron density maps based on these refinements reveal electron density in the center of the hormone binding domain in both data sets. In the case of 1,8-AEDANS, there is no density near Cys-10, but there is indication of a covalent link of 1,8-AEDANS to the e-amine of Lys-15, as was obtained in the crystal structures of N-bromoacetyl-hormone derivatives. Since the reactive acetyl group is the same for 1,5-AEDANS and 1,8-AEDANS, it is not clear why there is no involvement with Cys-10. Data for the ANS complex show density in the hormone binding site which is similar to that of 1,8-AEDANS. Higher resolution data for these complexes are needed to interpret changes in TTR conformation which may explain the mechanism of negative cooperativity.

Supported in part by DK-41009.