Human transthyretin in complex with iododiflunisal: structural features associated with a potent amyloid inhibitor

Abstract

Ex vivo and in vitro studies have revealed the remarkable amyloid inhibitory potency and specificity of iododiflunisal in relation to transthyretin [Almeida, Macedo, Cardoso, Alves, Valencia, Arsequell, Planas and Saraiva (2004) Biochem. J. 381, 351-356], a protein implicated in familial amyloidotic polyneuropathy. In the present paper, the crystal structure of transthyretin complexed with this diflunisal derivative is reported, which enables a detailed analysis of the protein-ligand interactions. Iododiflunisal binds very deep in the hormone-binding channel. The iodine substituent is tightly anchored into a pocket of the binding site and the fluorine atoms provide extra hydrophobic contacts with the protein. The carboxylate substituent is involved in an electrostatic interaction with the N(zeta) of a lysine residue. Moreover, ligand-induced conformational alterations in the side chain of some residues result in the formation of new intersubunit hydrogen bonds. All these new interactions, induced by iododiflunisal, increase the stability of the tetramer impairing the formation of amyloid fibrils. The crystal structure of this complex opens perspectives for the design of more specific and effective drugs for familial amyloidotic polyneuropathy patients.

Figure 1. Chemical structure of iododiflunisal, a difluorophenyl derivative of iodinated salicylic acid

Figure 2. C_α trace of TTR in complex with iododiflunisal

Monomers A (pink) and B (blue) form the crystallographic asymmetric unit. The 2-fold symmetry-related monomers A′ and B′ are displayed in grey and green respectively. (A) Omit F_o–F_c maps are contoured at 6σ over the hormone-binding site. The two strong density features found in each binding site correspond to the position of the iodines of the two symmetry-related iododiflunisal molecules. (B) Close view of the iododiflunisal binding site AA′ with omit maps contoured at 6σ (orange) and 3σ (red).

Figure 3. Stereo view of the interactions between the two symmetrically equivalent iododiflunisal molecules and TTR residues forming the AA′-binding site

Binding of iododiflunisal induces the rotation of the side chains of Ser¹¹⁷ and Ser¹¹⁷′ with the consequent formation of short hydrogen bonds between the hydroxy groups of Ser¹¹⁷ (A)–Ser¹¹⁷ (B) and Ser¹¹⁷ (A′)–Ser¹¹⁷ (B′). N^ζ of Lys¹⁵ is involved in weak hydrogen bond interactions with the CO₂H and the OH groups of the iodine-substituted ring. For the hydroxy group, the interaction is mediated by a water molecule. The iodine atom is involved in a series of hydrophobic contacts with the side chains of Thr¹⁰⁶, Ala¹⁰⁸, Thr¹¹⁹ and Val¹²¹.

Figure 4. Comparison of C_α–C_α distances between equivalent amino acids of the monomers forming each binding site (monomers AA′ and BB′) in apo-TTR and the TTR–iododiflunisal complex

The C_α–C_α distances (average across AA′ and BB′) for apo-TTR are plotted in black (d_apoTTR) and the differences between C_α–C_α distances in TTR–iododiflunisal and apo-TTR are plotted in white (d_complex–d_apoTTR).

Figure 5. The binding of iododiflunisal (A), flufenamic acid (B) and diclofenac (C) to TTR

The electrostatic surface representation of the AA′-binding site is presented along the x-axis. The two symmetry equivalent positions of the ligands are shown in orange and blue. The PDB accession numbers for the TTR–flufenamic acid and the TTR–diclofenac complexes are 1BM7 and 1DVX respectively. For clarity, the electrostatic surfaces of residues 15–17 and 109–110 (monomer A′) were removed since they are located in front of the binding molecules.