Acta Cryst. (2011). F67, 941–946doi:10.1107/S1744309111021099
Acta Crystallographica Section F
Expression, purification and preliminary
crystallographic analysis of human thyroid hormone
Wenzheng Zhang,aWei Peng,b
Mingzhuo Zhao,cDejun Lin,a
Zonghao Zeng,bWeihong Zhoua
and Mark Bartlama*
aTianjin Key Laboratory of Protein Science,
College of Life Sciences, Nankai University,
Tianjin 300071, People’s Republic of China,
bNational Laboratory of Biomacromolecules,
Institute of Biophysics (IBP), Chinese Academy
of Sciences, Beijing 100101, People’s Republic
of China, andcSchool of Physics, Hunan
University of Science and Technology,
Xiangtan 411201, People’s Republic of China
Received 10 March 2011
Accepted 1 June 2011
Thyroid hormone responsive protein (Thrsp, also known as Spot 14 and S14) is a
carbohydrate-inducible and thyroid-hormone-inducible nuclear protein specific
to liver, adipose and lactating mammary tissues. Thrsp functions to activate
genes encoding fatty-acid synthesis enzymes. Recent studies have shown that in
some cancers human Thrsp (hS14) localizes to the nucleus and is amplified,
suggesting that it plays a role in the regulation of lipogenic enzymes during
tumourigenesis. Thrsp, a member of the Spot 14 superfamily, is an acidic homo-
dimeric protein with no sequence similarity to other mammalian gene products
and its biochemical function is elusive. To shed light on the structure–function
relationship of this protein, human Thrsp was crystallized. Recombinant human
Thrsp (hThrsp), the N-terminally truncated human Thrsp10–146(hThrsp9) and
their selenomethionyl (SeMet) derivatives were expressed in Escherichia coli,
purified and crystallized using the hanging-drop vapour-diffusion method.
Diffraction-quality crystals were grown at 293 K using Li2SO4as a precipitant.
Using synchrotron radiation, data for the hThrsp SeMet derivative, hThrsp9 and
its SeMet derivative were collected to 4.0, 3.0 and 3.6 A˚resolution, respectively,
at 100 K. The crystals of full-length hThrsp and its SeMet derivative belonged to
space group P41212, with approximate unit-cell parameters a = b = 123.9,
c = 242.1 A˚, ? = ? = ? = 90.0?. In contrast, the crystals of the truncated hThrsp9
and its SeMet derivative belonged to space group P212121, with approximate
unit-cell parameters a = 91.6, b = 100.8, c = 193.7 A˚, ? = ? = ? = 90.0?. A
molecular-replacement solution calculated using a murine Spot 14 structure as
a search model indicated the presence of six molecules per asymmetric unit,
comprising three hThrsp homodimers.
The thyroid hormone responsive protein (Thrsp, also known as Spot
14 and S14) is an ?17 kDa acidic (pI 4.65) protein bearing no
sequence similarity to other functional motifs and with a strong
propensity for homodimerization (Cunningham et al., 1997; Chou et
al., 2007). It was first identified in 1982 as a result of its marked and
rapid induction by thyroid hormone (Seelig et al., 1982). The multi-
layered regulation and nuclear localization of Thrsp led researchers
to believe that it plays a role in tissue-specific control of metabolism
in response to changing dietary and hormonal factors (Campbell et
al., 2003; Cunningham et al., 1998; Franklyn et al., 1989). Thrsp has
been reported to be a homodimeric transcriptional activator that
serves as a component of a tripartite complex with a 36 kDa hepatic
protein in rat liver to modulate gene expression (Cunningham et al.,
1997). The biochemical mechanism of Thrsp is not known, but it
evidently functions to transduce hormone- and nutrient-related
signals to genes involved in lipid metabolism. Expression of the
human Thrsp (hThrsp, hS14) gene is abundant in those tissues that
are active in long-chain fatty-acid synthesis, such as the lactating
mammary gland (Dozin et al., 1986; Kinlaw et al., 1995; Ma &
Goodridge, 1992), and its localization in hepatic nuclei further
suggests that it functions in the regulation of lipogenic enzyme genes
(Brown et al., 1997; Cunningham et al., 1998; Kinlaw et al., 1992; Zhu
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HKL-2000 (Otwinowski & Minor, 1997). The native and SeMet-
derivative hThrsp9 crystals belonged to space group P212121, with
approximate unit-cell parameters a = 91.6, b = 100.8, c = 193.7 A˚,
? = ? = ? = 90.0?. Matthews coefficient analysis again suggested that
there were 12 molecules per asymmetric unit, with a VM(Matthews,
1968) of 2.35 A˚3Da?1and a solvent content of 48%. Data-collection
statistics are summarized in Table 1.
While we were in the process of further optimizing the crystals and
determining the structure by MAD/SAD phasing, the crystal struc-
ture of another member of the Spot 14 family, mouse S14 (PDB entry
3ont), was determined and reported by Colbert et al. (2010). Mouse
S14 was crystallized in space group I432 with a single monomer in the
asymmetric unit and a homodimer was inferred from crystallographic
symmetry. Given that the amino-acid sequence identity between
hThrsp and mouse S14 is 82%, we therefore attempted to determine
the crystal structure of hThrsp9 by the molecular-replacement
method using the structure of the mouse S14 monomer as a search
model. A molecular-replacement solution was found in Phaser with
six molecules per asymmetric unit comprising three homodimers and
corresponding to a Matthews coefficient VMof 4.70 A˚3Da?1and a
solvent content of 74%. An analysis of the molecular-replacement
solution showed loose packing of the hThrsp9 molecules (Fig. 5). This
solution is consistent with the self-rotation function calculated using
MOLREP, which shows threefold (? = 120?) and twofold (? = 180?)
axes but no substantial peaks in the ? = 60?section indicative of
sixfold symmetry (data not shown). After initial refinement of the
molecular-replacement solution, the working and free R factors were
reduced from 49.3% and 48.3% to 33.2% and 38.5%, respectively.
The unexpected high solvent content of 74% should help to
account for the consistently poor diffraction of hThrsp and hThrsp9
crystals. Although rare, there have been cases in which a structure has
been determined from crystals with unusually high solvent content.
For example, the diffraction by tetragonal crystals of human serum
albumin only reaches a maximum resolution of 3.0 A˚owing to their
high solvent content of 77% and loose packing in the crystal lattice
(Sugio et al., 1999). Further refinement of the hThrsp9 structure is
under way and will be reported elsewhere.
We would like to give special acknowledgement to Zihe Rao for
valuable guidance and we thank Xuemei Li, Wenlong Wang, Quan
Wang, Yi Han and Jianhui Li for technical instructions and assistance
with data collection. We also thank the staff members of KEK and
SSRF for assistance during data collection. This work was supported
20100470799) and the Ministry of Science and Technology ‘973’
Project (grant No. 2007CB914301).
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? Thyroid hormone responsive protein
Acta Cryst. (2011). F67, 941–946
Crystal packing for the molecular-replacement solution of hThrsp9 (viewed down
the threefold symmetry axis). The molecular-replacement solution of six hThrsp9
molecules (three homodimers) is shown in red. Symmetry-related molecules are
shown in green.