Structural and dynamics evidence for scaffold asymmetric flexibility of the human transthyretin tetramer.

Tadeo E Saldaño,Francesca Vallese,Sebastian Fernandez-Alberti,Rodolfo Berni,Giuseppe Zanotti,Alberto Ferrari,Ilaria Menozzi,Paola Berto

doi:10.1371/journal.pone.0187716

Abstract

The molecular symmetry of multimeric proteins is generally determined by using X-ray diffraction techniques, so that the basic question as to whether this symmetry is perfectly preserved for the same protein in solution remains open. In this work, human transthyretin (TTR), a homotetrameric plasma transport protein with two binding sites for the thyroid hormone thyroxine (T4), is considered as a case study. Based on the crystal structure of the TTR tetramer, a hypothetical D2 symmetry is inferred for the protein in solution, whose functional behavior reveals the presence of two markedly different Kd values for the two T4 binding sites. The latter property has been ascribed to an as yet uncharacterized negative binding cooperativity. A triple mutant form of human TTR (F87M/L110M/S117E TTR), which is monomeric in solution, crystallizes as a tetrameric protein and its structure has been determined. The exam of this and several other crystal forms of human TTR suggests that the TTR scaffold possesses a significant structural flexibility. In addition, TTR tetramer dynamics simulated using normal modes analysis exposes asymmetric vibrational patterns on both dimers and thermal fluctuations reveal small differences in size and flexibility for ligand cavities at each dimer-dimer interface. Such small structural differences between monomers can lead to significant functional differences on the TTR tetramer dynamics, a feature that may explain the functional heterogeneity of the T4 binding sites, which is partially overshadowed by the crystal state.

Highlights

Human transthyretin (TTR) is a homotetrameric protein involved in the transport in extracellular fluids of thyroxine (T4) and in the co-transport of vitamin A, by forming a macromolecular complex with plasma retinol-binding protein [1,2]
Out of a total of 240 human TTR structures present in the Protein Data Bank, 218 structures, including those of several TTR mutant forms and TTR-ligand complexes, belong to the orthorhombic space group P21212. In such structures a dimer is present in the asymmetric unit, and the second dimer is generated by symmetry, owing to the two-fold crystallographic axis coincident with the central channel in the TTR tetramer
Crystals of the structure presented here for the triple F87M/L110M/S117E TTR mutant belong to space group I222, where only one monomer is present in the asymmetric unit, and the tetramer is generated by the crystallographic symmetry (Fig 1)

Summary

Introduction

Human transthyretin (TTR) is a homotetrameric protein involved in the transport in extracellular fluids of thyroxine (T4) and in the co-transport of vitamin A, by forming a macromolecular complex with plasma retinol-binding protein [1,2]. Asymmetric flexibility of the human transthyretin tetramer form a very stable dimer through a net of H-bond interactions involving the two edge βstrands H and F, in such a way that a pseudo-continuous eight-stranded β-sandwich is generated, in which H and F β-strands from each monomer in the dimer are connected to each other by main-chain H-bonds and H-bonded water molecules. A long channel, coincident with one of the 2-fold symmetry axes, transverses the whole protein and harbors two T4 binding sites at the dimer-dimer interface

Methods

Results

Discussion

Conclusion