Crystal Structure Analysis Of Human Transthyretin Complexed With The Fluorescent Probe 1,5-AEDANS Shows Binding Differs From Spectral Data. Vivian Cody, Joseph R. Luft, Daniel Cotter, Colin Desnoes and Walter Pangborn, Hauptman-Woodward Medical Research Institute, Inc., Buffalo, NY 14203

Fluorescence spectral data for N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-AEDANS) binding to human transthyretin (hTTR) reveals covalent binding to Cys-10. However, structural data for the complex of hTTR and 1,5-AEDANS reveals no involvement of Cys-10, but does show interaction with Lys-15. Fluorecent probes are used to assess the hydrophobicity of protein binding sites and as a means of monitoring conformational changes in biological macromolecules. Fluorescence quenching studies of napthylsulfonic acid probes 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. Structure-activity data show that they can also act as competitive inhibitors for thyroxine (T₄) binding to transthyretin. In order to better understand negative cooperativity in hormone binding to TTR, we have carried out the X-ray crystal structure determination of hTTR co-crystallized with various fluorescent probes and report structural results for 1,5-AEDANS complex with hTTR and compare it to data for 1,8-AEDANS and 1,8-ANS. Crystals of the hTTR-1,5-AEDANS complex diffract to 1.9Å resolution and crystallize in the orthorhombic space group P2₁2₁2 with two independent monomers in the asymmetric unit. Refinement 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 both domains which differ from each other. In each domain there is no density near Cys-10, but there is indication of a covalent link of 1,5-AEDANS to the [epsilon]-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, as predicted from fluorecence spectral data. 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 Knoll Pharamaceuticals.