Photon Factory Activity Report 2016 #34 (2017)
BL-1A, BL-5A, BL-17A, AR-NW12A, AR-NE3A/ 2016G015, 2014G179

Crystal structures of eukaryotic and prokaryotic TRIC channels
Hanting Yang1 and Zhenfeng Liu1,*1

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics,
Chinese Academy of Sciences, Beijing 100101, China
1 Introduction
2+Ca signaling underlies a variety of fundamental
cellular activities including muscle contraction,neurotransmitter release, cell growth and apoptosis1.
Release of Ca2+ from the intracellular stores is supported
by a series of ion channels in sarcoplasma or endoplasma
reticulum (SR/ER). Among them, two isoforms of theTRimeric Intracellular Cation (TRIC) channel family,
named TRIC-A and B, modulate the release of Ca2+
through ryanodine receptor (RyR) or inositol triphosphate
receptor (IP3R), and maintain the homeostasis of ions
within SR/ER lumen2. As a SR/ER ion channel permeable
to monovalent cations, TRIC channels may account for
the activities of long-sought SR K+ channel3. TRIC-Achannel may regulate the Ca2+-release process throughRyR, and contributes to the maintenance of normal bloodpressure4. TRIC-B channel is believed to be involved inmodulating the Ca2+-release activity of IP3R and isessential for the perinatal lung development5, collagensynthesis and bone mineralization6. Mutations of the geneencoding TRIC-B (TMEM38B) in human are associatedwith a hereditary brittle bone disease (osteogenesisimperfecta)7,8. According to previous studies, TRIC
channels are crucial components for the Ca2+ signaling
and excitation-contraction coupling in eukaryotes, and theorthologs found in bacteria and archaea are likelyinvolved in K+ uptake in prokaryotes3,9.
Despite the important physiological functions of TRIC
channels, the lack of high-resolution structural
information has hindered elucidation of their porearchitectures and gating mechanisms. Here we present
structural studies on two eukaryotic TRIC-B channels(with/without Ca2+) from Caenorhabditis elegans(CeTRIC-B1/B2)10, as well as a prokaryotic TRIC
channel from Sulfolobus solfataricus (SsTRIC)11.
2 ExperimentProtein expression and purification
The genes encoding CeTRIC-B1/B2 were synthesized(Genscript) with optimized codon usage for protein
expression in Pichia pastoris. The target cDNA wasinserted into the pPICZ-A (or pPICZ-C for CeTRIC-B2)
vector. To improve stability of protein, 48 and 61 aminoacid residues at the flexible C-terminal regions ofCeTRIC-B1 and B2 were truncated, respectively.
CeTRIC-B1/B2 was expressed in P. pastoris GS115 strain
cells with 0.05% methanol for 48 h at 24℃. The cells
were harvested and the protein was extracted from themembrane by 1.5% Triton X-100.

The gene encoding SsTRIC protein was synthesized(GenScript) with optimized codon usage for protein
expression in Escherichia coli and inserted into pET21bvector. Expression of the SsTRIC protein was carried outin C41(DE3) cells with 1 mM IPTG for 2 h at 37℃. Thecells were harvested and the membrane protein was
solubilized by 1.5% dodecyl-β-D-maltoside (β-DDM).
Purification of the target proteins was achieved throughnickel/cobalt-affinity chromatography in solutions with ndecyl-β-D-maltopyranoside(DM). The eluted proteinfrom affinity-column was concentrated and further
purified by passing through the gel filtration column
Superdex 200 10/300 GL. The protein was concentrated
to 10-20 mg/ml for crystallization screening andoptimization.
Crystallization
The CeTRIC-B1 protein was initially crystallized
through hanging-drop vapor diffusion by mixing theprotein sample with a well solution containing 22%
PEG550 MME, 0.2 M NaCl, 0.1 M HEPES pH7.0, andthe CH3HgCl-derivated crystal in this plate form(in C2221
space group) was used for phasing. Another kind of
tetragonal-bipyramid crystals (in P41212 space group)from CeTRIC-B1 was grown with the reservoir solution
containing 20-24% PEG400, 10% glycerol, 50 mM ADA
buffer pH6.5, and the Ca2+/Rb+/Cs+- derivatized crystals
in this form were used for structure refinement andanalyses.
The CeTRIC-B2 protein was crystallized via hangingdrop vapor diffusion by mixing 1µl protein with 1 µl wellsolution containing 20% PEG400, 50 mM NaAc pH 4.4,
50 mM MgAc2 and 10 mM betaine hydrochloride.
The crystals of native and CH3HgCl/Tl+ derivatedSsTRIC were obtained through hanging-drops vapor
diffusion method by mixing 10 mg/ml protein with areservoir solution containing 22%-26% PEG 3000, 0.1 M
Tris-HCl pH 8.0, 0.2 M NaAc, 0.2 M KCl and 30%ethylene glycol at 1:1:0.2 (v:v:v) ratio.
Data collection and structure determination
Screening of crystals were mainly performed on BL1A,
BL5A, BL17A and NW12A beamlines at PF.
The anomalous diffraction data of Ca2+/Rb+/Cs+derivatized CeTRIC-B1 crystals were collected at BL1A
and BL5A. The datasets of CH3HgCl-derivatized
CeTRIC-B1, native CeTRIC-B2 and native SsTRIC werecollected at BL17U of Shanghai Synchrotron Radiation
Facility (SSRF). The data of Tl+-containing SsTRIC werePhoton Factory Activity Report 2016 #34 (2017)
collected at home source. For data processing, iMosflm orHKL2000 programs were used.
The initial experimental phase was solved by using theCH3HgCl-derivatized CeTRIC-B1 dataset through thesingle-wavelength anomalous diffraction (SAD) methodby using the Autosol program of the Phenix suite. The
structural model of CeTRIC-B1 was completed throughiterative manual model building in COOT and refinementin CNS program. The structure of CeTRIC-B2 weresolved through molecular replacement.
For SsTRIC, the structure was solved by SAD withanomalous diffraction dataset of Hg-labeled A15C crystal.
The structures were refined by using CNS, Phenix andRefmac programs.

conformations, a mechanistic model has been proposed todemonstrate the intriguing gating mechanism of
eukaryotic TRIC channels10.
For SsTRIC, the monomer-monomer interfaces within
the trimer were stabilized by hydrophobic interactions
between the protein and a putative triacylglycerol
molecule instead of a PIP2 molecule. Meanwhile, thecytoplasmic vestibule of SsTRIC is occluded by a pluglike motif in the loop region between the fifth and sixthtransmembrane helices. The plug-like motif binds to thepore lumen in a position resembling that of the PIP2 headgroup in CeTRIC-B1/B2. Based on biochemical andelectrophysiological data, a mechanistic gating model of
SsTRIC channel has been proposed11.
In conclusion, structural and functional analyses on
CeTRIC-B1/B2 and SsTRIC provide a unified framework
for understanding the gating mechanism of bothprokaryotic and eukaryotic TRIC channels.
Acknowledgement

Fig. 1 Structures of TRIC channels. a, The
homotrimeric CeTRIC-B1 channel in complex withphosphatidylinositol 4,5-biphosphate (PIP2) lipidmolecules. b, The trimeric SsTRIC channel in complex
with triacylglycerol. The protein subunits are presented asribbon models, the PIP2 and triacylglycerol molecules areshown as yellow-and-red sphere models. Both structuresare viewed along the membrane plane.

We thank PF staff members for their technical support
during crystal screening and data collection. This research
project is financially supported by funding from National
973 project, Chinese Academy of Sciences and the"National Thousand Young Talents" program from theoffice of Global Experts Recruitment in China.
References
13 Results and Discussion
We have solved the structures of CeTRIC-B1 andCeTRIC-B2 channels at two different conformationalstates (with/without Ca2+-bound) at 3.3 and 2.3 Åresolution respectively, and the structure of SsTRIC
channel at 2.2 Å resolution. Our data demonstrated thatboth eukaryotic and prokaryotic TRIC channels form
symmetrical homotrimer with a novel asymmetrical ionpermeating pathway within each monomer. The center ofthe hourglass-shaped pores are flanked by funnel-likevestibules on cytoplasmic and luminal sides of eukaryotic
TRIC channels, or on cytoplasmic and extracellular sidesof prokaryotic TRIC channels. The pore center may
contain a monovalent cation-binding site likely
contributing to the ion selectivity of the TRIC channels.
At the same time, we also discovered several striking
features about TRIC channels from structural views. For
the CeTRIC-B channels, TRIC-B1/B2 proteinsspecifically bind an endogenous lipid named
phosphatidylinositol 4,5-biphosphate (PIP2). The PIP2
molecule mediates trimerization of TRIC channels,
contributes directly to the formation of pore architecture,and interacts with the putative voltage-sensing motif aswell as a Ca2+-binding loop region. While the finding of a
PIP2 molecule in the intracellular ion channel isunprecedented, it has expanded our current knowledge
about the role of PIP2 in the function of ion channels.
Furthermore, by comparing the Ca2+-bound and Ca2+-free

2

3456

Michael J. Berridge, P. L. M. D. B. The
versatility and universality of calcium signalling.
Nature Reviews Molecular Cell Biology, 11,
doi:10.1038/35036035 (2000).
Yazawa, M. et al. TRIC channels are essential
for Ca2+ handling in intracellular stores. Nature448, 78-82, doi:10.1038/nature05928 (2007).
Takeshima, H., Venturi, E. & Sitsapesan, R. Newand
notableion-channels
inthesarcoplasmic/endoplasmic reticulum: do they
support the process of intracellular Ca2+release?
The
Journalofphysiology,
doi:10.1113/jphysiol.2014.281881 (2014).
Tao, S. et al. Facilitated hyperpolarization
signaling in vascular smooth muscleoverexpressing TRIC-A channels. The Journal of
biological chemistry 288, 15581-15589,
doi:10.1074/jbc.M112.435396 (2013).
Yamazaki, D. et al. Essential role of the TRIC-B
channel in Ca2+ handling of alveolar epithelial
cells and in perinatal lung maturation.
Development136,2355-2361,
doi:10.1242/dev.036798 (2009).
Cabral, W. A. et al. Absence of the ER Cation
ChannelTMEM38B/TRIC-B
Disrupts
Intracellular
Calcium
Homeostasis
andDysregulates Collagen Synthesis in Recessive
Osteogenesis Imperfecta. PLOS Genetics 12,
e1006156,
doi:10.1371/journal.pgen.1006156(2016).

Photon Factory Activity Report 2016 #34 (2017)
7891011

Volodarsky, M. et al. A deletion mutation inTMEM38B associated with autosomal recessive
osteogenesis imperfecta. Human mutation 34,582-586, doi:10.1002/humu.22274 (2013).
Lv, F. et al. Two novel mutations in TMEM38B
result in rare autosomal recessive osteogenesis
imperfecta. J Hum Genet 61, 539-545,doi:10.1038/jhg.2016.11 (2016).
Silverio, A. L. F. & Saier, M. H. Bioinformatic
Characterization of the Trimeric Intracellular
Cation-Specific Channel Protein Family. The
Journal of Membrane Biology 241, 77,
doi:10.1007/s00232-011-9364-8 (2011).
Yang, H. et al. Pore architecture of TRIC
channels and insights into their gatingmechanism.
Nature538,537-541,
doi:10.1038/nature19767 (2016).
Ou, X. et al. Ion- and water-binding sites inside
an occluded hourglass pore of a trimeric
intracellular cation (TRIC) channel. BMC Biol15, 31, doi:10.1186/s12915-017-0372-8 (2017).

* liuzf@sun5.ibp.ac.cn