Journal of Biological Chemistry
Volume 285, Issue 41, 8 October 2010, Pages 31731-31741
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Protein Structure and Folding
Novel Zn2+-binding Sites in Human Transthyretin: IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION*

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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.

Amyloid
NMR
Vitamin A
X-ray Crystallography
Zinc
Retinol-binding Protein
Transthyretin

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The atomic coordinates and structure factors (codes 3GRG, 3GRB, 3GPS, and 3DGD) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported by CNPq (INCT-Bioimagem e Biologia Estrutural), CAPES Nanobiotec-Brasil 04-2008, and FAPERJ (to D. F. and L. M. T. R. L.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S6.

1

Both authors contributed equally to this work.

2

Supported by a fellowship from CNPq, CAPES PNPD-2009, and FAPERJ.