Novel Zn2+-binding Sites in Human Transthyretin: IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION*

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn²⁺ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn²⁺ at pH 4.6–7.5. All four structures reveal three tetra-coordinated Zn²⁺-binding sites (ZBS 1–3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn²⁺ binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn²⁺. Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn²⁺. HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn²⁺ binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn²⁺, which is consistent with the tertiary structural perturbation provoked by Zn²⁺ binding, tetramer stability is only marginally affected by Zn²⁺. These data highlight structural and functional roles of Zn²⁺ in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.