Photon Factory Activity Report 2019 #37 (2020)

BL-1A, BL-5A, BL-17A, AR-NE3A, AR-NW12A/2016G508

Structural biology of human TRPV1 channel intracellular domain
toward elucidation of redox mechanism

Miki Tanaka1,2, Kaori HAYAKAWA1,2, Nozomi OGAWA3, Tatsuki KUROKAWA4, Yasuo
MORI3 and Masaki UNNO1,2,*
1 Graduate School of Science and Engineering, Ibaraki University,
4-1-12 Nakanarusawa, Hitachi, 316-8511, Japan
2 Frontier Research Center for Applied Atomic Sciences,162-1 Shirakata, Tokai, Naka 319-1106, Japan
3 Graduate School of Engieering, Kyoto University, 8-13 Katsura, Kyoto 615-8510
4 Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hazamacho, Yufu 879-5593, Japan
1 Introduction
Transient Receptor Potential (TRP) cation channel
exists as a tetramer and it is involved in various
biological functions as sensors of stimulus reception.
One of them, TRPV1, is activated by capsaicin - main
component of pepper -, heat higher than 42°C, andoxidation. TRPV1 has a very characteristic structure
called Ankyrin Repeat Domain (ARD) at the Nterminus. Human TRPV1 (hTRPV1) forms an intersubunit disulfide bond (Cys258- Cys742), suggestingthat it is involved in redox-sensing [1]. Therefore, inthis study, we aimed to determine the structure ofhTRPV1-ARD containing Cys258, the structure ofhTRPV1-Ctrem containing Cys742 and their complex
under non-reducing condition to elucidate the binding
mode of the disulfide bond at atomic level.
2 ExperimenthTRPV1-ARD (101-365) fused with GST-tag wasexpressed in E.coli Rosetta (DE3). After GST affinity
chromatography was performed to collect the GSThTRPV1-ARD, the GST-tag was cleaved byThrombin. Thereafter, GST affinity chromatography
was performed again to separate GST-tag andsucceeded obtaining hTRPV1-ARD. Crystallization of
the purified hTRPV1-ARD was performed with thesimilar conditions as rTRPV1-ARD [2]. Because
hTRPV1-Ctrem was not able to be obtained with
sufficient quantity, a peptide of small region ofhTRPV1-Ctrem was prepared and co-crystallization
with hTRPV1-ARD was attempted. X-ray diffraction
experiments and the structural analysis wereperformed.
3 Results and Discussion
Good quality crystals were grown in the presenceof 0.125 M sodium citrate (pH 5.0), 5% (w/v) PEG
8000 and 2.5% (v/v) glycerol as a reservoir solution.
In the X-ray diffraction experiments, crystals did notdiffract X-ray well at the initial stage and data suitable
for structure analysis could not be obtained, however
4.5 Å resolution data could be obtained under thecondition that the cryoprotectant was changed toglucose from glycerol. Molecular replacement wasperformed using the structure of rTRPV1-ARD as an
initial model, and the structure of hTRPV1-ARD could
be determined at the main chain level (Fig. 1). This
crystal belonged to the space group C2, has a highsolvent content of 72%, and has a very uniquestructure containing six molecules of hTRPV1-ARD in
the asymmetric unit. When compared with thestructure of rTRPV1-ARD, some differences in thestructure of the ligand binding pocket were found near
Cys258 which was suggested to form a disulfide bondwith the C-terminal Cys742. This difference between
structures of hTRPV1-ARD and rTRPV1-ARD can beconsidered to relate to the high oxidation sensitivityof Cys258 in hTRPV1 [3].

Fig. 1: Overall Structure of hTRPV1-ARD [3].
Acknowledgement
The X-ray data collection was also conducted at theSwiss Light Source in the summer 2019 (Proposalnumbers 20191094 and 20191134 at PSI). This work
was partly supported by the Ibaraki Prefecture
Sendouteki Kenkyu. This work was also performed
under the Cooperative Research Program of"Network Joint Research Center for Materials andDevices.".
References[1] N. Ogawa et al., J. Biol. Chem. 291, 4197 (2016).
[2] P. V. Lishko et al,, Neuron 54, 905 (2007).
[3] M. Tanaka et al., Acta Crystallogr. F76, 130 (2020)
Research Achievements
1. Poster Award in the 46th Symposium on
Biomolecular Science (2019), Tsukuba

Photon Factory Activity Report 2019 #37 (2020)

2.

Flash and Poster Presentations
AsCA2019, Singapore* masaki.unno.19@vc.ibaraki.ac.jp

Award,