Hydrogen-bond network and pH sensitivity in human transthyretin.

Takeshi Yokoyama,Katsuhiro Kusaka,Takaaki Hosoya,Takashi Ohhara,Yuko Nabeshima,Ichiro Tanaka,Kazuo Kurihara,Taro Yamada,Nobuo Niimura,Mineyuki Mizuguchi

doi:10.1107/s090904951302075x

Abstract

Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm(3) for four months. The neutron crystal structure solved at 2.0 Å revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

Highlights

Amyloidosis refers to a variety of conditions wherein normally soluble proteins become insoluble and are deposited in the extracellular space of various organs or tissues, causing damage
TTR misfolding and aggregation are associated with amyloid diseases such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy (Rochet & Lansbury, 2000; Buxbaum & Tagoe, 2000; Kelly, 1996; Benson, 1989)
Large TTR crystals with a volume of 2.5 mm3 were obtained and the neutron crystal structure was solved at 2.0 Aresolution using iBIX

Summary

Introduction

Amyloidosis refers to a variety of conditions wherein normally soluble proteins become insoluble and are deposited in the extracellular space of various organs or tissues, causing damage. Transthyretin (TTR) is a tetrameric protein and transports hormone thyroxine and retinol A in the blood. TTR misfolding and aggregation are associated with amyloid diseases such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy (Rochet & Lansbury, 2000; Buxbaum & Tagoe, 2000; Kelly, 1996; Benson, 1989). TTR is a tetramer of 55 kDa composed of four identical polypeptide chains (subunits A–D) of 127 amino acid residues (Fig. 1). Each polypeptide chain forms eight -strands and one -helix. The intersubunit contacts are divided roughly into monomer–monomer interactions and dimer–dimer

Methods

Results

Conclusion