1: IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography.

Proc Natl Acad Sci U S A.

2017 May 2;114(18):4661-4666. doi: 10.1073/pnas.1701420114. Epub 2017 Apr 17.

Kozo Hamada, Hideyuki Miyatake, Akiko Terauchi, and Katsuhiko Mikoshiba

Operating principle of the IP3 receptor elucidated by X-ray crystallography

Calcium ions (Ca2+) are essential for cellular function. In particular, the IP3 receptor, which acts as a gate regulating Ca2+ release from the cell's Ca2+ store (the endoplasmic reticulum), is key to this function. Elucidating its working mechanism was a pressing issue. We succeeded in crystallizing the large protein of the cytoplasmic domain of the IP3 receptor, which consists of 2,217 amino acid residues, both in the presence and absence of IP3. Using X-ray crystallography at the synchrotron radiation facility (SPring-8), we successfully elucidated the operating principle of the IP3 receptor (see figure). The process is as follows: ① IP3 binds to its binding site, ② causing a conformational change in the helical domain. ③ A unique leaflet-like structure in the region near the channel moves outward, transmitting the conformational change to the channel. ④ The α-helix of the channel pore moves, opening the pore (the "faucet"), and Ca2+ is released into the cytoplasm. This corrects the previous claim from cryo-electron microscopy analysis that the IP3 binding site and the C-terminal region of the channel bind directly to open the channel.

The IP3 receptor is related to endoplasmic reticulum stress, autophagy, and apoptosis, and is involved in neurodegenerative diseases such as spinocerebellar ataxia, dementia, and exocrine disorders. If compounds that act on the newly identified leaflet structure can be discovered, it is expected to serve as a new drug discovery target for the treatment and prevention of many diseases.

For receptors and channels, the upper limit for crystallization was typically a length of 600 amino acid residues, but we succeeded in crystallizing a massive protein of 2,217 amino acid residues.

The crystallization of this massive protein is a world record for receptors and channels. We grew crystals in the cold room of the Mikoshiba Laboratory, sent them to the large synchrotron radiation facility (SPring-8) to obtain and analyze diffraction patterns, and used the results to search for even better conditions. The key to our success was the ability to successfully introduce mutations to suppress structural fluctuations. This was the result of 15 years of patient research to find the right conditions.

We also determined the sequence of the IP3 receptor in the protozoan *Trypanosoma cruzi*, the pathogen that causes Chagas disease, and found it to be essential for the protozoan's survival. Despite low homology in the overall sequence, the leaflet structure was highly conserved. This suggests it has important significance for life phenomena and could also be a target for drug discovery.

RIKEN Brain Science Institute

Senior Team Leader, Laboratory for Developmental Neurobiology

Katsuhiko Mikoshiba (48th)