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diffraction structural biology
834 doi:10.1107/S090904951302075X J. Synchrotron Rad. (2013). 20, 834–837
Journal of
ISSN 0909-0495
Received 20 May 2013
Accepted 25 July 2013
Hydrogen-bond network and pH sensitivity in
human transthyretin
Takeshi Yokoyama,a* Mineyuki Mizuguchi,a Yuko Nabeshima,a Katsuhiro Kusaka,b
Taro Yamada,b Takaaki Hosoya,b,c Takashi Ohhara,d Kazuo Kurihara,e
Ichiro Tanakab,c and Nobuo Niimurab
aFaculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0914,
Japan, bFrontier Research Center for Applied Atomic Sciences, Ibaraki University, 162-1 Shirakata,
Tokai, Ibaraki 319-1106, Japan, cCollege of Engineering, Ibaraki University, 4-12-1 NakaNarusawa, Hitachi, Ibaraki 316-8511, Japan, dResearch Center for Neutron Science and
Technology, Comprehensive Research Organization for Science and Society, 162-1 Shirakata,
Tokai, Ibaraki 319-1106, Japan, and eQuantum Beam Science Directorate, Japan Atomic Energy
Agency, 2-4 Shirakata, Tokai, Ibaraki 319-1195, Japan. E-mail: tyokoya3@pha.u-toyama.ac.jp
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 timeof-flight method. Crystals for the neutron diffraction experiments were grown
up to 2.5 mm3 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
Keywords: neutron protein crystallography; transthyretin; amyloidosis;
hydrogen-bond network; pH sensitivity.
1. 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
interactions. The monomer–monomer interactions are formed
between subunits A and B or C and D, whereas the dimer–
dimer interactions are formed between subunits A and D or B
and C. These contacts are important for the stability of the
TTR tetramer. The mechanism underlying TTR amyloidogenesis in humans is the subject of intense investigation. Ratelimiting dissociation of the tetramer into its component
subunits is necessary. The folded subunits must also undergo
partial denaturation to produce an amyloidogenic intermediate, a step that is often linked thermodynamically to
dissociation. This intermediate then misassembles into
numerous morphologies including amorphous aggregates and
spherical aggregates, and ultimately into amyloid fibrils
(Lashuel et al., 1998, 1999; Colon & Kelly, 1992). While the
acidic conditions greatly accelerate the rate-limiting TTR
dissociation and the aggregate formation, some small molecules, that bind to TTR, kinetically stabilize TTR and suppress
the amyloid fibril formation (Bulawa et al., 2012; Hurshman et
al., 2004; Liu et al., 2000; Kelly et al., 1997; Klabunde et al.,
2000). Recent structural studies have begun to reveal the
structural changes by the lowered pH in both wild-type and
amyloidogenic mutant TTR. The crystal structure of the I84A
amyloidogenic mutant showed notable conformational
changes at pH 4.6 compared with that of the I84A structure
determined at pH 7.5. In these structures a large conformational change is found at the EF-helix and loop (Pasquato et
al., 2007). Furthermore, the crystal structure of the wild-type
TTR determined at pH 4.0 and 3.5 also showed conformational changes in the same region (Palaninathan et al., 2008).
Although many X-ray crystal structures of TTR have been
solved so far, the precise molecular mechanisms underlying
TTR aggregation remain elusive. The pH-dependent effects in
proteins are mainly electrostatic in nature and originate from
changes in the protonation states of acidic and basic residues
(Yang & Honig, 1993). To further investigate the structural
explanation for the pH-dependent effect of TTR, detailed
information on the hydrogen and protonation states is needed.
The neutron protein crystallography is preferred as a tool to
determine the hydrogen bonding, the protonation states and
the hydration of macromolecules, since the neutron-scattering
lengths of hydrogen and deuterium are comparable with those
of other elements (Niimura & Bau, 2008). We report here the
neutron crystallographic analysis of TTR (Yokoyama et al.,
2012). The neutron crystal structure solved at 2.0 Å provides
the protonation states and detailed information about the
hydrogen bonds. We discuss the origin of pH sensitivity
related to the structural stability of TTR.
2. Materials and methods
2.1. Protein preparation and crystallization
In order to obtain a large crystal suitable for neutron
crystallography, an N-terminal truncated TTR lacking 1–11
was expressed (Yokoyama et al., 2012). The expression and the
purification of N-terminal truncated TTR were carried out as
previously described (Miyata et al., 2010). The purified protein
was concentrated up to 19 mg ml1 and frozen with liquid
nitrogen until use. As N-terminal truncated TTR was likely to
crystallize only from the magnesium-ion-containing solutions
as a result of the many crystallization screenings, the crystallization screenings were carried out again using protein solution supplemented with 0.2 M MgCl2. Single crystals were
observed using tri-ammonium citrate pH 7.0 as the precipitating agent within a few days. In order to avoid neutron
incoherent scattering from H atoms, the crystals for the
neutron diffraction experiments were grown using the protein
solution exchanged by heavy water and precipitating agents
prepared with the heavy water. The large crystal of N-terminal
truncated TTR was obtained in a drop containing 1.85 M triammonium citrate pD 7.4 and 0.4 M MgCl2 at 293 K in four
months by the sitting-drop vapour-diffusion method (Fig. 2).
2.2. Neutron diffraction experiments and structure
Crystals were mounted in quartz capillaries with the reservoir solution to avoid dryness and then the capillaries were
sealed with wax. Time-of-flight neutron diffraction data were
collected in the BL-03 iBIX installed at the pulsed neutron
source of MLF in J-PARC (Tanaka et al., 2010). The diffraction data sets were collected at room temperature using 13
detectors placed at 2center from 33.0
 to 139.4 (Hosoya et al.,
2009). To complete the data, 41 data sets (41 crystal orientations from one crystal) were collected using a wavelength
range from 2.7 Å to 6.7 Å with a crystal-to-detector distance of
490 mm. The exposure times were 22 h for each set at 120 kW
J-PARC accelerator power and 12 h at 220 kW. The diffraction
peaks were observed distinctly (Fig. 3). The collected data
were indexed, integrated and scaled with STARGazer which
was developed to process iBIX time-of-flight diffraction data
(Ohhara et al., 2009). The X-ray crystal structure of TTR at
room temperature was used as the initial model (Protein Data
Bank ID: 3u2i). The structure was refined using PHENIXREFINE for neutron structure refinement with several stepwise cycles of manual model building using COOT (Adams et
al., 2011; Emsley & Cowtan, 2004). The data collection and
refinement statistics are listed in Table 1.
3. Results and discussion
3.1. Hydrogen-bond network and pH sensitivity
The neutron structure of TTR was identical to the X-ray
structure of TTR with a root-mean-square deviation of 0.34 Å
diffraction structural biology
J. Synchrotron Rad. (2013). 20, 834–837 Takeshi Yokoyama et al.  Human transthyretin 835
Figure 2
The TTR crystal for the neutron diffraction experiment.
Figure 1
Structure of the TTR tetramer. The dimer–dimer contacts are indicated
as dashed circles. Subunit A is shown in grey, B in green, C in blue and
D in magenta.
between the C atoms of the two structures. First, the protonation states of the histidine residues were determined based
on the Fourier peaks at the positions of hydrogen (deuterium)
atoms of the difference Fourier map omitting each histidine
residue. The Fourier density showed that His31 was doubly
protonated, whereas His56, His88 and His90 were singly
protonated. Furthermore, the orientations of the water
molecules were determined by the same method. Fourteen out
of 55 water molecules observed in the asymmetric unit were
identified as complete water (D2O but neither DO nor O).
Among the four histidine residues, the protonation state of
His88 is very interesting. The unprotonated N1 atom of His88
accepted a hydrogen bond from the water molecule (Fig. 4).
This hydrogen bond was involved in a large hydrogen-bond
network consisting of Thr75, Trp79, His88, Ser112, Pro113,
Thr118(B) and four water molecules. As this hydrogen-bond
network was made up of ten hydrogen bonds, it is important
for the structural stability of TTR. It is suggested that the
double protonation of His88 may break this hydrogen-bond
network and destabilize the TTR monomer structure. This
network is also involved in the dimer–dimer interaction, which
is important for the tetramer formation of TTR (Fig. 4). This
interaction includes the two hydrogen bonds formed between
Ser112 of subunit A and Ser112 of D and between Tyr114 of
subunit A and Ala19 of D. These hydrogen bonds appeared to
be important in light of the fact that Ser112Ile and Tyr114His
mutants are amyloidogenic variants (Murakami et al., 1994;
Shinohara et al., 2003). This dimer–dimer arrangement is
stabilized by the hydrogen-bond network of His88 stabilizing
the GH-loop (Pro113 and Tyr114) (Fig. 4). As Ser112 and
Pro113 are members of the hydrogen-bond network, the full
protonation of His88 by acidic condition probably breaks this
hydrogen-bond network and destabilizes the tetramer. In
order to determine the residues responsible for the pH
sensitivity of TTR, the neutron structure of TTR was
compared with the X-ray structure at pH 4.0 (Palaninathan et
al., 2008). The conformational changes of Asp74, His88 and
Glu89 were observed by structural comparison. Asp74 forms
a hydrogen bond with Ser77 at neutral pH, whereas it forms
a hydrogen bond with a water molecule on the molecular
surface at pH 4.0. His88 is involved in the large hydrogenbond network at neutral pH but swings away into the solvent
without any hydrogen bonds with the water molecules at pH
4.0. Glu89 forms a salt bridge with Lys76 at neutral pH,
whereas the salt bridge is broken at pH 4.0. These results
suggest that Asp74, His88 and Glu89 are mainly responsible
for the pH sensitivity of TTR. Among these residues, His88
involves the large hydrogen-bond network composed of ten
hydrogen bonds. Therefore, His88 is likely predominant in pH
diffraction structural biology
836 Takeshi Yokoyama et al.  Human transthyretin J. Synchrotron Rad. (2013). 20, 834–837
Table 1
Statistics on the data collection and refinement.
Numbers in parentheses refer to the highest-resolution shell.
Crystal data
Resolution range (Å) 12.1–2.0 (2.07–2.00)
Space group P21212
Unit cell (Å) a = 44.3, b = 86.4, c = 66.7
Unique reflections 15307 (2080)
Rsym (%)† 19.1 (30.6)
Completeness (%) 86.4 (72.5)
I/ 4.3 (1.5)
Redundancy 2.6 (1.7)
Refinement data
Rfactor (%)‡ 23.4
Rfree (%)§ 27.2
RMSD bonds (Å) 0.010
RMSD angles () 1.207
† Rsym = hkli|Ii(hkl)  I( h kl)|/hkliIi(hkl). ‡ Rfactor = |Fo|  |Fc|/|Fo|, where Fo
and Fc are the observed and calculated structure factor amplitudes, respectively. § Rfree
was calculated with 5% of the data excluded from the refinement. Figure 4
The hydrogen-bond network consisted of His88 and water molecules. The
|Fo| |Fc| difference neutron scattering length density map was calculated
omitting His88 and water molecules (contoured at 2.5). The hydrogen
bonds are indicated as dashed yellow lines. The residue names of subunit
A are shown in grey and those of D are in magenta. Unexchangeable
H atoms are not displayed.
Figure 3
Time-of-flight neutron diffraction image recorded by iBIX. The threedimensional diffraction data were projected in time-of-flight.
3.2. CH  O hydrogen bond
Careful inspection of the H atoms affords some clues to
understanding the CH  O weak hydrogen bonds. The close
CH  O contacts are thought to play an important role in the
stabilization and function of biological molecules. CH  O
contacts are increasingly being accepted as genuine hydrogen
bonds (Desiraju & Steiner, 1999; Wahl & Sundaralingam,
1997). According to the energies calculated by Jiang & Lai
(2002), the CH  O hydrogen bond has a binding strength of
1.9 kJ mol1 and an optimum C  O distance of 3.3 Å,
whereas the conventional hydrogen bond has a binding energy
of 5.5 kJ mol1 and an optimum O  O (N) distance of
2.8 Å. The list of hydrogen bonds and possible CH  O
hydrogen bonds formed in the dimer–dimer contact (subunits
A and D) are summarized (Table 2). Not only three hydrogen
bonds but also eight CH  O hydrogen bonds are formed in
this region. A simple calculation of the energies underscores
the importance of CH  O hydrogen bonds, because the
energy sum of these bonds is comparable with that of
conventional hydrogen bonds and is not negligible. At this
interface, Tyr114 seems to be a major contributor to the
intersubunit contact. The substitution of Tyr114 with His,
which is known as an amyloidogenic variant, perturbed the
stability of the quaternary structure. These results suggest that
CH  O hydrogen bonds may play an important role in
stabilizing the quaternary structure of TTR.
4. Conclusions
Large TTR crystals with a volume of 2.5 mm3 were obtained
and the neutron crystal structure was solved at 2.0 Å resolution using iBIX. The neutron structure revealed that the
protonation state of His88 is closely related to tetramer
stability. The mechanisms underlying accelerated amyloid
fibril formation by acidic conditions were structurally
explained by neutron protein crystallography. Although it is
difficult to obtain crystals large enough for neutron diffraction
experiments, these results comfirmed the usefulness of
neutron protein crystallography.
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diffraction structural biology
J. Synchrotron Rad. (2013). 20, 834–837 Takeshi Yokoyama et al.  Human transthyretin 837
Table 2
List of hydrogen bonds and possible CH  O hydrogen bonds formed
between subunits A and D.
Acceptor Donor Distance (Å)
Hydrogen bonds O  O, N (O  D)
A19(A)–O Y114(D)–D 3.0 (2.2)
S112(A)–O S112(D)–D 2.5 (1.9)
A19(D)–O Y114(A)–D 3.0 (2.2)
CH  O hydrogen bonds O  C (O  H)
A19(A)–O Y114(D)–H2 3.4 (2.6)
V20(A)–O P113(D)–H2 3.6 (2.8)
V20(A)–O Y114(D)–H1 3.4 (2.8)
A19(A)–O S112(D)–H3 3.6 (2.9)
A19(D)–O S112(A)–H3 3.6 (2.9)
V20(D)–O P113(A)–H2 3.6 (2.7)
A19(D)–O Y114(A)–H2 3.4 (2.6)
V20(D)–O Y114(A)–H1 3.4 (2.8)

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